MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Analytical Biochemistry' source. Fri, 19 Mar 2010 17:17:21 +0100 http://www.medworm.com/index.php?rid=3371010&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20230769%26dopt%3DAbstract Authors: Mercedes-Camacho AY, Etzkorn FA Peptidyl prolyl cis-trans isomerase (PPIase) interacting with NIMA-1 (Pin1) catalyzes the cis-trans isomerization of pSer/pThr-Pro amide bonds. Pin1 is a two-domain protein that represents a promising target for the treatment of cancer. Both domains of Pin1 bind the pSer/pThr-Pro motif; PPIase enzymatic activity occurs in the catalytic domain, and the WW domain acts as a recognition module for the pSer/pThr-Pro motif. An assay we call an Enzyme-Linked Enzyme Binding Assay (ELEBA), was developed to measure the K(d) of ligands that bind selectively to the WW domain. A ligand specific for the WW domain of Pin1 was covalently immobilized in a 96-well plate. Commercially available Pin1 conjugated to horseradish peroxidase was used for chemiluminescen... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3371010 Fri, 12 Mar 2010 00:00:00 +0100 3371010 http://www.medworm.com/index.php?rid=3371009&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20230770%26dopt%3DAbstract Authors: Safavi A, Farjami F The composite film based on Nafion and hydrophilic room-temperature ionic liquid (RTIL) 1-butyl-3-methyl-imidazolium chloride ([bmim]Cl) was used as an immobilization matrix to entrap myoglobin (Mb). The study of IL-Mb interaction by UV-Vis spectroscopy showed that Mb retains its native conformation in the presence of IL. The immobilized Mb displayed a pair of well-defined cyclic voltammetric peak with a formal potential (E(o)) of -0.35 V in a 0.1 M phosphate buffer solution (PBS) of pH 7.0. The immobilized Mb exhibited excellent electrocatalytic response to the reduction of hydrogen peroxide, based on which a mediator free amperometric biosensor for hydrogen peroxide was designed. The linear range for the determination of hydrogen peroxide was from 1.0- 18... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3371009 Fri, 12 Mar 2010 00:00:00 +0100 3371009 http://www.medworm.com/index.php?rid=3371008&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20230771%26dopt%3DAbstract Authors: Takahashi H, Matsumoto A, Sugiyama S, Kobori T Prevailing conventional microbial detection methods largely depend upon microbial cultivation in selective media which requires several days. PCR-based methods, including quantitative reverse transcription PCR, are also rapid and useful methods for identification of target microbes although, for practical use, they still suffer from disadvantages such as contamination problems and cost. Herein we demonstrate that RNA-primed rolling circle amplification (RPRCA) using phi29 DNA polymerase, a precircularized probe and SYBR Green II, achieved real-time detection of specific mRNA from living microbes. The precircularized DNA probe was prepared by intramolecular ligation using CircLigase and treated by exonuclease I to eliminate uncircu... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3371008 Fri, 12 Mar 2010 00:00:00 +0100 3371008 http://www.medworm.com/index.php?rid=3371007&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20230772%26dopt%3DAbstract We described a visible dye-based staining method for DNA in polyacrylamide gels using ethyl violet (EV). The novel method is a background-free, sensitive, economical and simple procedure involving only staining and washing steps that can be completed within 30 min. As little as 0.8-1.6 ng of phiX174 DNA/HaeIII can be detected by EV, which is about eight-fold more sensitive than that of nile blue (NB) stain and two-fold less sensitive than that of ethidium bromide (EB) stain. PMID: 20230772 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3371007 Fri, 12 Mar 2010 00:00:00 +0100 3371007 http://www.medworm.com/index.php?rid=3371006&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20230773%26dopt%3DAbstract Authors: Liao Y, Yuan R, Chai Y, Zhuo Y, Yang X In this work, protonated L-cysteine was entrapped in Nafion (Nf) membrane by cation-exchange function, forming Nafion-Cys composite membrane, which was more stable, compact, biocompatible and favorable for mass and electron transfer compared with Nf film solely. Then gold nanoparticles were adsorbed onto the electrode surface by thiol groups on the composite membrane. After that, nano-Au monolayer was formed, onto which carcinoembryonic antibody (anti-CEA) was loaded to prepare carcinoembryonic antigen (CEA) immunosensor. The results indicated that the immunosensor had good current response for CEA, using potassium ferricyanide as the redox probe. A linear concentration range of 0.01 to 100 ng/mL with a detection limit of 3.3 pg/mL (S/N =... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3371006 Fri, 12 Mar 2010 00:00:00 +0100 3371006 http://www.medworm.com/index.php?rid=3371005&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20230774%26dopt%3DAbstract We describe the development of a novel method for the assay of serine/threonine protein kinases based on fluorescence lifetime. The assay consists of three generic peptides (which have been used by others in the assay of over 140 protein kinases in various assay formats) labeled with a long lifetime fluorescent dye (14 or 17 ns), which act as substrates for protein kinases, and an iron(III) chelate which modulates the fluorescence lifetime of the peptide only when it is phosphorylated. The decrease in average fluorescence lifetime as measured in a recently developed fluorescence lifetime plate reader (Edinburgh Instruments) is a measure of the degree of phosphorylation of the peptide. We present data showing the assay performs as well as and in some cases better than the 'gold standard' ra... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3371005 Fri, 12 Mar 2010 00:00:00 +0100 3371005 http://www.medworm.com/index.php?rid=3371004&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20230775%26dopt%3DAbstract Authors: Watanabe T, Enomoto T, Takahashi M, Honma S, Honma KI, Ohmiya Y We constructed a multi-channel perfusion culture system. Using this device, we developed a perfusion device to detect six samples at the same time and demonstrated the response to dexamethasone of the circadian-related promoter activity in Rat1 fibroblast cells. We could detect the sequential phase shifts of the circadian peaks that are dependent on the timing of the drug treatments. We also demonstrate a temporal dual-reporter assay using two kinds of secreted luciferase in the perfusion culture. The combination of secreted luciferase and multiple perfusion culture assay system reveals the effects of transient drug treatment for the pharmacological assay. PMID: 20230775 [PubMed - as supplied by publisher] (So... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3371004 Fri, 12 Mar 2010 00:00:00 +0100 3371004 http://www.medworm.com/index.php?rid=3371013&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20227379%26dopt%3DAbstract Authors: Ibáñez G, McBean JL, Astudillo YM, Luo M Epigenetic regulation through protein posttranslational modifications is essential in development and disease. Among the key chemical modifications is protein methylation carried out by protein methyltransferases (PMTs). Quantitative and sensitive PMT-activity assays can provide valuable tools to investigate PMT functions. Here we developed an enzyme-coupled luminescence assay for S-adenosyl-L-methionine(AdoMet/SAM)-based PMTs. In this assay, S-adenosyl-L-homocystine (AdoHcy/SAH), the byproduct of PMT-involved methyation, is sequentially converted to adenine, adenosine monophosphate, and then adenosine triphosphate (ATP) by 5'-methylthio-adenosine/AdoHcy nucleosidase (MTAN), adenine phosphoribosyl transferases (APRT), and pyru... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3371013 Thu, 11 Mar 2010 00:00:00 +0100 3371013 http://www.medworm.com/index.php?rid=3371012&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20227380%26dopt%3DAbstract This study shows that purified Wnt3a, Wnt7a, and Wnt5a have different binding specificities for Fzds and SFRPs. PMID: 20227380 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3371012 Thu, 11 Mar 2010 00:00:00 +0100 3371012 http://www.medworm.com/index.php?rid=3371011&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20227381%26dopt%3DAbstract In this study we present an efficient phosphorylation-free and ligase-free PCR-based multiple site-directed mutagenesis which allows simultaneous mutations up to six distal-sites. This method could be extended to any plasmid DNA which is isolated from dam(+) Escherichia coli strains, and the results showed that the simultaneously mutagenic efficiencies of quadruple-mutation and sextuple-mutation were up to 80 and 40%, respectively. PMID: 20227381 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3371011 Thu, 11 Mar 2010 00:00:00 +0100 3371011 http://www.medworm.com/index.php?rid=3371017&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20226156%26dopt%3DAbstract Authors: Arsene CG, Henrion A, Diekmann N, Manolopoulou J, Bidlingmaier M Inter-assay variation of antibody based routine tests is hampering comparability of measurement results for growth hormone (GH) between different laboratories and decision making in clinical practice. Here it is demonstrated, that quantification of GH by isotope dilution mass spectrometry (IDMS) constitutes a way to precise and reliable results which can be referred to in evaluation of performance of commercial test kits. With the IDMS method developed, tryptic cleavage products YSFLQNPQTSLCFSESIPTPSNR (T6) and LEDGSPR (T12) of GH are quantified by LC/MS-MS using the isotopically labeled forms of the peptides as internal standards. The GH cleavage fragments are obtained by whole-serum tryptic proteolysis and then... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3371017 Tue, 09 Mar 2010 00:00:00 +0100 3371017 http://www.medworm.com/index.php?rid=3371016&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20226157%26dopt%3DAbstract In this study, we applied relative quantitative proteomic analysis based on label-free multiple reaction monitoring (MRM) to discover biological changes of adipokines under oxidative stress. Among a total of 194 identified proteins, 8 proteins were selected and quantified between control and hydrogen peroxide (H(2)O(2))-treated groups by label-free MRM quantification. Secretion levels of matrix metalloproteinase-2 (MMP-2), stromal cell-derived factor-1 (SDF-1, CXCL12), resistin, and complement factor D (CFD, adipsin) decreased, while the secretion levels of tissue inhibitor of metalloproteinase-2 (TIMP-2) and aldolase A increased. Here, we suggest that our study with label-free quantitative analysis will contribute to the efficient quantitative analysis of targeted proteins in complex mixt... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3371016 Tue, 09 Mar 2010 00:00:00 +0100 3371016 http://www.medworm.com/index.php?rid=3371015&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20226158%26dopt%3DAbstract Authors: Lian W, Wu D, Lim DV, Jin S Using a newly developed fluorescent nanoparticle (NP) which gives rise to a high intensity and stable fluorescent light, a sensitive antibody microarray assay system was developed for specific detection of bioterrorism agents, as exemplified by Ricin, cholera toxin (CT) and staphylococcal enterotoxin B (SEB). The antibody microarray uses a sandwich format, which consists of capture antibodies, analytes (toxins), biotinylated detection antibodies, and avidin conjugated NP. In all three cases, polyclonal antibodies displayed superiority over monoclonal antibodies in capturing toxins on microarray slides, even when the polyclonal and monoclonal antibodies had similar affinity as determined by ELISA. The detection system was successfully used to detect ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3371015 Tue, 09 Mar 2010 00:00:00 +0100 3371015 http://www.medworm.com/index.php?rid=3371014&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20226159%26dopt%3DAbstract Authors: Sieber MW, Recknagel P, Glaser F, Witte OW, Bauer M, Claus RA, Frahm C Reverse transcription followed by quantitative PCR (rt-qPCR) has become the state of the art tool for quantification of nucleic acids. However, there are still significant problems associated with its sensitivity, reproducibility, efficiency and the choice of an appropriate rt-qPCR kit. The purpose of this study is to give insights into strategies to optimize and validate the performance of currently available kits for rt-qPCR and to provide up-to-date information about the benefits, potentials and pitfalls of rt-qPCR assays. A selection of 9 cDNA synthesis and 12 qPCR kits were tested using samples obtained from three species (mouse, rat and human) and three transcripts (Gapdh, Actb and Hmbs) under highly ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3371014 Tue, 09 Mar 2010 00:00:00 +0100 3371014 http://www.medworm.com/index.php?rid=3354670&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20211145%26dopt%3DAbstract Authors: Laremore TN, Ly M, Solakyildirim K, Zagorevski DV, Linhardt RJ Separation of milligram amounts of heparin oligosaccharides ranging in degree of polymerization from 4 to 32 is achieved within 6 hours using continuous-elution polyacrylamide gel electrophoresis (CE-PAGE) on commercially available equipment. The purity and structural integrity of CE-PAGE-separated oligosaccharides are confirmed by strong-anion exchange high-pressure liquid chromatography, electrospray ionization Fourier transform mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy. The described method is straightforward and time-efficient, affording size-homogeneous oligosaccharides that can be used in sequencing, protein binding, and other structure-function relationship studies. PM... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3354670 Fri, 05 Mar 2010 00:00:00 +0100 3354670 http://www.medworm.com/index.php?rid=3354669&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20211590%26dopt%3DAbstract We report a high-throughput two-dimensional microarray-based screen, incorporating both target binding intensity and off-rate, which can be used to analyze thousands of compounds in a single binding assay. Relative binding intensities and time-resolved dissociation is measured for labeled tumor necrosis factor alpha (TNF-alpha) bound to a peptide microarray. The time-resolved dissociation is fitted to a one-component exponential decay model, from which relative dissociation rates are determined for all peptides with binding intensities above background. We show that most peptides with the slowest off-rates on the microarray also have the slowest off-rates when measured by surface plasmon resonance (SPR). PMID: 20211590 [PubMed - as supplied by publisher] (Source: Analytical Biochemistr... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3354669 Thu, 04 Mar 2010 00:00:00 +0100 3354669 http://www.medworm.com/index.php?rid=3354668&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20211591%26dopt%3DAbstract Authors: Meyer H, Liebeke M, Lalk M Systems biology studies assume the acquisition of reliable and reproducible data sets. Metabolomics, in particular, requires comprehensive evaluated workflows to enable the analysis of hundreds of different compounds. Therefore, a protocol to elucidate the metabolome of the Gram-positive pathogen, Staphylococcus aureus COL strain, grown in a chemically defined medium is introduced here. Different standard operating procedures in the field of metabolome experiments were tested for common pitfalls. These included: suitable and fast sampling processes, efficient metabolite extraction, quenching effectiveness (energy charge) and estimation of leakage and recovery of metabolites. Moreover, a cell disruption protocol for S. aureus was developed and optimiz... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3354668 Thu, 04 Mar 2010 00:00:00 +0100 3354668 http://www.medworm.com/index.php?rid=3354667&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20211592%26dopt%3DAbstract Authors: Zhu X, Gu Y A novel adsorbent of carboxymethyl-beta-cyclodextrin (CM-beta-CD/TiO(2)) modified nanometer TiO(2) was prepared and used as a solid phase extraction (SPE) material coupled to fluorescence spectroscopy determination of L-tryptophan (L-Trp) in biological samples. The experimental conditions for modified nanometer TiO(2) separation/preconcentration of L-Trp were optimized. The adsorption capacity of CM-beta-CD/TiO(2) for L-Trp was 75.2 mug/g. The linear range, detection limit (DL) and the relative standard deviation (R.S.D.) was 0.10 approximately 1.20 mug/mL, 18.8 ng/mL, and 0.67% (n =3, 1.0 mug/mL) respectively, with a preconcentration factor of 10. The developed method was applied to determination of L-Trp in real sample and the recoveries were found to be in the r... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3354667 Thu, 04 Mar 2010 00:00:00 +0100 3354667 http://www.medworm.com/index.php?rid=3347116&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20206593%26dopt%3DAbstract Authors: Zimmerman LB, Lee KD, Meyerhoff ME The preparation of DNA-tagged liposomes containing an encapsulated prosthetic group tracer, pyrroloquinoline quinone (PQQ), and their application to the development of a sandwich type hybridization assay for the visual detection of single stranded DNA, is described. Capture DNA is conjugated to the surface of microtiter plate wells through a biotin-streptavidin interaction. Target DNA is incubated with the plate in high salt concentrations. The reporter DNA-tagged liposomes encapsulating PQQ, the prosthetic group of the apo-enzyme glucose dehydrogenase (GDH), are used as the label to probe for bound target DNA. After washing away unbound liposomes and subsequent lysis of the bound fraction by surfactant, PQQ is released and available to activ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3347116 Wed, 03 Mar 2010 00:00:00 +0100 3347116 http://www.medworm.com/index.php?rid=3347123&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20206114%26dopt%3DAbstract Authors: Schuck P For the detailed analysis of sedimentation velocity data, the consideration of radial-dependent baseline offsets is indispensable. Two main approaches are data differencing ('delta-c' approach), and explicit inclusion of baseline parameters ('direct boundary model' of the raw data). The present work aims to clarify the relationships between the two approaches. To this end, a simple model problem is examined. We show that the explicit consideration of the baseline in the model is equivalent to a differencing scheme where from all data points their average value is subtracted. Pair-wise differencing in the 'delta-c' approach always results in higher parameter uncertainty. For equidistant time points, the increase is smallest when the reference points are taken at interv... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3347123 Tue, 02 Mar 2010 00:00:00 +0100 3347123 http://www.medworm.com/index.php?rid=3347122&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20206115%26dopt%3DAbstract Authors: Romero-Calvo I, Ocón B, Martínez-Moya P, Suárez MD, Zarzuelo A, Martínez-Augustin O, Medina FS It is becoming standard practice to measure a housekeeping gene, typically actin, in Western blots, as it is the rule in RNA blots. We have applied reversible Ponceau staining to check equal loading of gels and measured actin in parallel under different conditions. Our results show that densitometric analysis is comparable with both techniques. Therefore routine quantitation of Ponceau staining before antibody probing is validated as an alternative to actin blotting. PMID: 20206115 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3347122 Tue, 02 Mar 2010 00:00:00 +0100 3347122 http://www.medworm.com/index.php?rid=3347121&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20206116%26dopt%3DAbstract Authors: Moaddel R, Bighi F, Yamaguchi R, Patel S, Ravichandran S, Wainer IW Membranes from stably transfected cell lines that expresses two point mutations of the human organic cation 1 transporter (hOCT1), R488M and G465R, have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form the Cellular Membrane Affinity Chromatography (CMAC) (hOCT1(G465R)) and CMAC(hOCT1(R488M)). Columns were created using both stationary phases and frontal displacement chromatography experiments were conducted using [(3)H]-methyl phenyl pyridinium, [(3)H]-MPP(+), as the marker ligand and various displacers, including the single enantiomers of verapamil, fenoterol and isoproterenol. The chromatographic data obtained was used to refine a previously develo... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3347121 Tue, 02 Mar 2010 00:00:00 +0100 3347121 http://www.medworm.com/index.php?rid=3347120&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20206117%26dopt%3DAbstract Authors: Tummala M, Lee SM, Chess E, Hu P Pertussis toxoid, an acellular pertussis vaccine prepared by hydrogen peroxide treatment in the presence of Fe(3+), has not been well characterized. Since the toxoid has been a part of the DTaP vaccine for infants, it is of interest and significance to have a clear understanding of its structure. The five subunits of pertussis toxin (PT) have a combined molecular weight of approximately 95,000. The peroxide treatment in toxoid formation introduces additional complexity into the protein sequence. To maximize sequence coverage, a 2D LC-MS/MS approach was used to analyze the tryptic digest of toxoid as a whole. An analytical scale HPLC using a pentafluorophenyl (PFP) column was used as the first dimensional LC for fraction collection. The fraction... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3347120 Tue, 02 Mar 2010 00:00:00 +0100 3347120 http://www.medworm.com/index.php?rid=3347119&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20206118%26dopt%3DAbstract Authors: León K, Riverón AM, Arencibia O, López-Cánovas L We standardized the zinc-imidazole negative staining method for detecting chromosomal-sized DNA molecules separated by Pulsed Field Minigel Electrophoresis. The best experimental conditions were: separating large DNA molecules in minigels of 0.4cm in thickness, further incubating it with 40mM ZnSO(4) solution and finally with 0.1M and 2M imidazole solutions, successively. The lowest yeast cells/miniplug useful in DNA band detection was 3x10(7)cells, as occurred with ethidium bromide stained minigel. Electrophoresis patterns were visualized as colorless bands contrasting against a white background after illuminating the minigel with white light. This negative staining method is nontoxic and preserves chemical ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3347119 Tue, 02 Mar 2010 00:00:00 +0100 3347119 http://www.medworm.com/index.php?rid=3347118&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20206119%26dopt%3DAbstract We describe, for the first time, reliable detection, visualization, and quantification of mature cathepsin K to femtomole resolution using gelatin zymography. The specificity of the method was validated with cathepsin K knockdown using siRNA transfection of human monocyte derived macrophages, and enzymatic activity confirmed with Z-GPR-AMC substrate hydrolysis was fit to a computational model of enzyme kinetics. Further, cathepsin K zymography was used to show that murine osteoclasts secrete more cathepsin K than stored intracellularly, which was opposite to the behavior of the macrophages from which they were differentiated. In summary, this inexpensive, species-independent, antibody-free protocol describes a sensitive method with broad potential to elucidate previously undetectable cathe... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3347118 Tue, 02 Mar 2010 00:00:00 +0100 3347118 http://www.medworm.com/index.php?rid=3347117&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20206120%26dopt%3DAbstract Authors: Men J, Lang L, Wang C, Wu J, Zhao Y, Jia PY, Wei W, Wang Y This work aimed to evaluate a method to detect the residual ricin in animal tissues. Immunoprecipitation and Sandwich ELISA were used to detect ricin in the tissues of intoxicated mice. The monoclonal antibodies (Mab) 4C13 and 3D74 were used to assay the whole ricin molecules via Sandwich ELISA. Mab 4C13 was conjugated with Sepharose 4B to capture ricin or ricin A chain (RTA) by immunopreciptation. Mice i.v. injected with ricin at the dosage of 5 mug/mouse were killed at different time-points after intoxication. The serum, liver, kidney, lung and intestine were harvested. High levels of ricin were found in serum and liver samples at each poisoning time point by Sandwich ELISA, suggested the possibility of determining r... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3347117 Tue, 02 Mar 2010 00:00:00 +0100 3347117 http://www.medworm.com/index.php?rid=3336039&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20197057%26dopt%3DAbstract In conclusion, one should consider the role of direct excitation of ANS and Bis-ANS at 280 nm to ensure a proper interpretation of fluorescence signals resulting from dye - protein interactions. When ANS or Bis-ANS are to be used for protein characterization, we recommend to selectively excite the dyes at the higher absorption wavelength maximum (370 nm or 385 nm, respectively). PMID: 20197057 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3336039 Sat, 27 Feb 2010 00:00:00 +0100 3336039 http://www.medworm.com/index.php?rid=3336038&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20197058%26dopt%3DAbstract Authors: Ye Y, Stivers JT We have developed the first economical and rapid nonradioactive assay method that is suitable for high-throughput screening of the important pharmacological target human DNA (cytosine-5)-methyltransferase I (DNMT1). The method combines three key innovations: the use of a truncated form of the enzyme that is highly active on a 26 base pair hemimethylated DNA duplex substrate, the introduction of the methylation site into the recognition sequence of a restriction endonuclease, and the use of a fluorogenic readout method. The extent of DNMT1 methylation is reflected in the protection of the DNA substrate from endonuclease cleavage, which would otherwise result in a large fluorescence increase. The assay has been validated in a high-throughput format and trivial c... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3336038 Sat, 27 Feb 2010 00:00:00 +0100 3336038 http://www.medworm.com/index.php?rid=3328050&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20193653%26dopt%3DAbstract We present a method for isolating microsomal-type membranes from small amounts of Arabidopsis thaliana plant material that does not rely on ultracentrifugation, but instead uses the lower relative centrifugal force (21,000g) of a microcentrifuge. We show that the 21,000g pellet is equivalent to that obtained at 100,000g, and that it contains all the membrane fractions expected in a conventional microsomal fraction. Our method incorporates specific manipulation of sample density throughout the procedure, with minimal pre-clearance, minimal volumes of extraction buffer and minimal sedimentation pathlength. These features allow maximal membrane yields, enabling membrane isolation from limited amounts of material. We further demonstrate that conventional ultracentrifuge-based protocols give su... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3328050 Fri, 26 Feb 2010 00:00:00 +0100 3328050 http://www.medworm.com/index.php?rid=3328052&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20188690%26dopt%3DAbstract Authors: Ständker L, Zachgo V, Hillemanns P, Rösinger M, Forssmann WG, Hass R A fragment of the human beta chain of hemoglobin (hHEMbeta111-146) showed broad antimicrobial properties. The 3.9 kDa peptide was postulated to occur in high concentrations in placenta tissue. We established a reliable method to quantify hHEMbeta111-146 in placenta tissue. Our methodology comprises a tissue extraction step (1), a chromatographic enrichment (2) and a final quantification step (3) by ELISA. The specifity of the ELISA reaction was confirmed by parallel analysis of the samples via Western blot (step 4). The ELISA measured the absorbance of a tetramethylbenzidine substrate at 450 nm. It showed no cross-reactivity with the corresponding gamma - and alpha - HEM regions and a low cross reac... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3328052 Wed, 24 Feb 2010 00:00:00 +0100 3328052 http://www.medworm.com/index.php?rid=3328051&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20188691%26dopt%3DAbstract Authors: Choulier L, Shvadchak VV, Naidoo A, Klymchenko AS, Mély Y, Altschuh D A ratiometric fluorescent sensor was obtained by solid-phase synthesis of a peptide singly labeled at its N-terminus with a 3-hydroxychromone (3HC) derivative, an environmentally sensitive fluorophore with a two-band emission. The construct contains the binding site recognized by an antibody fragment, svFv1F4(Q34S) with nM affinity. The dye only marginally affected the kinetic and equilibrium binding parameters of the scFv-peptide interaction, as measured by surface plasmon resonance. On interaction with the antibody fragment, the sensor showed up to 47% change in the ratio of its two emission bands, indicating an enhanced screening of the 3HC fluorophore from bulk water. Competition with two unlabeled ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3328051 Wed, 24 Feb 2010 00:00:00 +0100 3328051 http://www.medworm.com/index.php?rid=3328054&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20188057%26dopt%3DAbstract Authors: Groeve MR, Tran GH, Hoorebeke AV, Stout J, Desmet T, Savvides SN, Soetaert W Glycoside phosphorylases (GPs) are interesting enzymes for the glycosylation of chemical molecules. They only require a glycosyl phosphate as sugar donor and an acceptor molecule with a free hydroxyl group. Their narrow substrate specificity, however, limits the application of GPs for general glycoside synthesis. Although an enzyme's substrate specificity can be altered and broadened by protein engineering and directed evolution, this requires a suitable screening assay. Such a screening assay has not yet been described for GPs. Here, we report a screening procedure for GPs based on the measurement of released inorganic phosphate in the direction of glycoside synthesis. It appeared necessary to inhibi... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3328054 Tue, 23 Feb 2010 00:00:00 +0100 3328054 http://www.medworm.com/index.php?rid=3328053&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20188058%26dopt%3DAbstract Authors: Choi YS, Pennington CL, Wood TD Many neuropeptides lack suitable amino acid residues for modification by existing selective isotope labeling methods and use in relative quantitation by mass spectrometry. In order to address this issue, a new stable isotope labeling method which targets tyrosine residues by coupling with light (d(0)) or heavy cysteine (d(2)) in the presence of tyrosinase was developed. Optimal derivatization conditions for 1 muM of leucine-enkephalin were achieved when 10 mM of cysteine and 200 U/mL of tyrosinase at pH 6.8-7.2 were used for a 60 minute incubation period at room temperature. Under these conditions, leucine-enkephalin present at concentrations as low as 125 nM was successfully labeled. When comparisons between the lightly labeled (d(0)) and heavi... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3328053 Tue, 23 Feb 2010 00:00:00 +0100 3328053 http://www.medworm.com/index.php?rid=3315595&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20184856%26dopt%3DAbstract Authors: Faggiano S, Ronda L, Bruno S, Jankevics H, Mozzarelli A Dynamic light scattering (DLS) is a technique capable of determining the hydrodynamic radius of proteins. From this parameter, a molecular weight can be assessed, provided that an appropriate calibration curve is available. To this goal, a globin-based calibration curve was used to determine the polymerization state of a recombinant hemoglobin-based oxygen carrier and to assess the equivalent molecular weight of hemoglobins conjugated with polyethylene glycol molecules. The good agreement between DLS values and those obtained from gel filtration chromatography is a consequence of the high similarity in structure, shape and density within the globin superfamily. Moreover, globins and heme proteins in general share similar ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3315595 Mon, 22 Feb 2010 00:00:00 +0100 3315595 http://www.medworm.com/index.php?rid=3315594&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20184857%26dopt%3DAbstract Authors: Sobhi HF, Minkler PE, Hoppel CL The measurement of acyl-CoA dehydrogenase activities is an essential part of the investigation of patients with suspected defects in fatty acid oxidation. Multiple methods are available for the synthesis of the substrates used for measuring acyl-CoA dehydrogenase activities; however the yields are low and the products are used without purification. Additionally, the reported characterization of acyl-CoAs focuses on the CoA moiety, not the acyl group. Herein, we describe the synthesis of three medium-chain acyl-CoAs from mixed anhydrides of the fatty acids using an aqueous-organic solvent mixture optimized to obtain the highest yield. First, cis-4-decenoic acid and 2,6-dimethylheptanoic acid were prepared (3-phenylpropionic acid is commerically a... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3315594 Mon, 22 Feb 2010 00:00:00 +0100 3315594 http://www.medworm.com/index.php?rid=3311874&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20178770%26dopt%3DAbstract We describe a novel cell capture assay that enables several candidate antibodies to be evaluated quickly as to their relative binding efficacies to their cell-surface targets. We used chimeric rituximab and murine anti-CD20 monoclonal antibodies as cell capture agents on a functionalized microscope slide surface to assess their relative binding affinities based on how well they capture CD20-expressing mammalian cells. We found that these antibodies' concentration-dependent cell capture profiles correlate with their relative binding affinities. A key observation of this assay involved understanding how differences in capture surfaces affect the assay results. This approach can find utility when an antibody or antibody fragment against a known cell line needs to be selected for targeting stu... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3311874 Sat, 20 Feb 2010 00:00:00 +0100 3311874 http://www.medworm.com/index.php?rid=3311873&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20178771%26dopt%3DAbstract We present an optimized and validated liquid chromatography-electrospray ionization tandem mass spectrometric (LC-ESI-MS/MS) method for the simultaneous measurement of concentrations of different ceramide species in biological samples. The method of analysis of tissue samples is based on Bligh and Dyer extraction, reverse-phase HPLC separation and multiple reaction monitoring of ceramides. Preparation of plasma samples also requires isolation of sphingolipids by silica gel column chromatography prior to LC-ESI-MS/MS analysis. The limits of detection and quantification are in a range of 5-50 pg/ml for distinct ceramides. The method was reliable for inter-assay and intra-assay precision, accuracy and linearity. Recovery of ceramide subspecies from human plasma, rat liver and muscle tissue we... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3311873 Sat, 20 Feb 2010 00:00:00 +0100 3311873 http://www.medworm.com/index.php?rid=3311872&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20178772%26dopt%3DAbstract Authors: Samejima K, Hiramatsu K, Takahashi K, Kawakita M, Kobayashi M, Tsumoto H, Kohda K A method for the quantification of acetylpolyamines, N(1),N(12)-diacetylspermine (DiAcSpm), monoacetylspermidine (AcSpd), and N(1),N(8)-diacetylspermidine (DiAcSpd), identifying each compound simultaneously, was developed with the goal of evaluating these acetylpolyamines as potential biomarkers of cancer. The method consists of prepurification of acetylpolyamines in urine with commercially available cartridges and derivatization with heptafluorobutyric (HFB) anhydride. HFB derivatives of acetylpolyamines were simultaneously determined using (15)N-labeled acetylpolyamines as internal standards by ESI-TOF MS. After the method was validated, the urinary acetylpolyamines of 38 cancer patients were q... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3311872 Sat, 20 Feb 2010 00:00:00 +0100 3311872 http://www.medworm.com/index.php?rid=3302962&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20175981%26dopt%3DAbstract Authors: Ducros V, Pollicand M, Laporte F, Favier A Plasma vitamin K1 (phylloquinone) determination is commonly used for the diagnosis of vitamin K deficiency in patients suffering from lipid malabsorption. Moreover current evidence that adequate vitamin K intake, and correspondingly adequate plasma vitamin K1 concentration, could be also of importance in relation to bone and brain diseases emphasizes the need to improve the current analytical methods. We developed a liquid chromatography coupled to tandem mass spectrometry method using a stable isotope D4-ring labeled internal standard of vitamin K1, and operating in the multiple reaction monitoring mode by the selection of a precursor and product ions. Atmospheric pressure chemical ionization (APCI) method was shown to be more sensit... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3302962 Fri, 19 Feb 2010 00:00:00 +0100 3302962 http://www.medworm.com/index.php?rid=3302961&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20175982%26dopt%3DAbstract We describe a simple and rapid method for determining the critical micelle concentration (CMC) of surfactants from fiber-optic measurements of refractive index. The refractive index of an aqueous surfactant solution was monitored, as the surfactant concentration was increased using an automated dispensing system. Upon reaching the surfactant's CMC value, an abrupt change was observed in the rate of increase of the refractive index with increasing concentration. The measurement system provides rapid semi-automatic data collection and analysis, increasing the precision, sensitivity, and range of applicability of the technique while substantially decreasing the amount of manual intervention required. Measurements of CMC for sodium dodecylsulfate (8.10 mM), cetyltrimethylammonium chloride (1.5... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3302961 Fri, 19 Feb 2010 00:00:00 +0100 3302961 http://www.medworm.com/index.php?rid=3302960&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20175983%26dopt%3DAbstract In conclusion, this study suggests that the procedure described here can be widely used to refold GPCRs expressed as inclusion bodies in E. coli. PMID: 20175983 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3302960 Fri, 19 Feb 2010 00:00:00 +0100 3302960 http://www.medworm.com/index.php?rid=3302959&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20175984%26dopt%3DAbstract Authors: Büchele B, Zugmaier W, Lunov O, Syrovets T, Merfort I, Simmet T Sesquiterpene lactones such as helenalin have generally been considered as highly promising compounds for the treatment of inflammatory disorders. Although sesquiterpene lactones are known to inhibit signaling through transcription factor NF-kappaB, the nature of their molecular targets remains controversial. To characterize the interactions of helenalin with putative target proteins, a surface plasmon resonance-based method was developed and validated to analyze the interactions of helenalin with the NF-kappaB protein p65/RelA, with recombinant IkappaB kinase (IKK) alpha and beta, and the intracellular antioxidant, glutathione, all immobilized on sensor chips. At pH 7.4; helenalin is interacting with RelA (K... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3302959 Fri, 19 Feb 2010 00:00:00 +0100 3302959 http://www.medworm.com/index.php?rid=3302958&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20175985%26dopt%3DAbstract Authors: Munoz L, Selig R, Yeung YT, Peifer C, Hauser D, Laufer S Development of inhibitors that target inactive kinase conformations is becoming a more attractive approach to kinase inhibitor research. The major advantage of this methodology is that targeting the inactive conformation reduces competition with high intracellular ATP concentrations. p38alpha MAPK signalling has been identified as the principal mediator of inflammation associated with a spectrum of disorders (e.g. arthritis, Alzheimer's disease, various malignancies). To allow identification and development of p38alpha MAPK inhibitors that preferentially bind to the inactive conformation, a novel fluorescence polarisation-based binding assay is presented. The assay is homogeneous, requires low amounts of the kinase and f... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3302958 Fri, 19 Feb 2010 00:00:00 +0100 3302958 http://www.medworm.com/index.php?rid=3291302&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20167203%26dopt%3DAbstract Authors: Uthus EO A capillary electrophoresis (CE) method for the determination of methionine sulfoxide reductase A and methionine sulfoxide reductase B activities in mouse liver is described. The method is based on detection of the 4-(dimethylamino)azobenzene-4'-sulfonyl derivative of L-methionine (dabsyl Met), the product of the enzymatic reactions when either dabsyl L-methionine S-sulfoxide or dabsyl L-methioinine R-sulfoxide are used as substrates. The method provides baseline resolution of the substrates and it therefore can be used to easily determine the purity of the substrates. The method is rapid (approximately 20 min sample to sample), requires no column regeneration, and uses very small amounts of buffers. Separation was performed by using a 75 mum i.d. polyimide coated-fus... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3291302 Mon, 15 Feb 2010 00:00:00 +0100 3291302 http://www.medworm.com/index.php?rid=3284585&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20159005%26dopt%3DAbstract Authors: Yin BC, Zuo P, Huo H, Zhong X, Ye BC Gold nanoparticles can be exploited to facilitate a highly sensitive and selective metal ions detection based on fluorescence anisotropy assay with metal ion-dependent DNA-cleaving DNAzyme. This assay allows rapid and accurate determination of metal ions in aqueous media at room temperature. The method has been demonstrated for determination of Cu(2+) and Pb(2+) ions. The detection sensitivity can be significantly improved to 1 nM by using a "nanoparticle enhancement" approach. Moreover, the assay was also tested in 384-well plates for high-throughput routine determination of toxic metal ions in environmental samples. The method show distinct advantages over conventional methods in terms of its potential sensitivity, specificity and ability... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3284585 Sat, 13 Feb 2010 00:00:00 +0100 3284585 http://www.medworm.com/index.php?rid=3284584&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20159006%26dopt%3DAbstract Authors: Goodpaster AM, Romick-Rosendale LE, Kennedy MA Use of NMR-based metabonomics to search for human disease biomarkers is becoming increasingly common. For many researchers, the ultimate goal is translation from biomarker discovery to clinical application. Studies typically involve investigators from diverse educational and training backgrounds, including physicians, academic researchers, and clinical staff. In evaluating potential biomarkers, clinicians routinely use statistical significance testing language, whereas academicians typically use multivariate statistical analysis techniques that do not perform statistical significance evaluation. In this paper, we outline an approach to integrate statistical significance testing with conventional principal components analysis data ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3284584 Sat, 13 Feb 2010 00:00:00 +0100 3284584 http://www.medworm.com/index.php?rid=3284590&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20156414%26dopt%3DAbstract Authors: Calles-Enríquez M, Ladero V, Fernández M, Martín MC, Alvarez MA This work describes a simple method for extracting high-quality microbial RNA from fermented milk. Pre-treatment consists merely of adding a concentrated solution of sodium citrate to the milk product. The proposed method provides consistently better yields of high-quality RNA than the conventional method, independent of the texture of the fermented milk. Further, it can be used with small amounts of starting material, requiring only the use of Eppendorf-size centrifuges -a great advantage for analytical laboratories. The RNA obtained is suitable for the detection of live microorganisms and for transcriptional studies based on RT-qPCR. PMID: 20156414 [PubMed - as supplied by publisher] (Source: ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3284590 Fri, 12 Feb 2010 00:00:00 +0100 3284590 http://www.medworm.com/index.php?rid=3284589&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20156415%26dopt%3DAbstract Authors: Murugaiah V, Zedalis W, Lavine G, Charisse K, Manoharan M Gene silencing induced by short interfering RNA (siRNA) has proven useful in genomic research and has great potential for therapeutic applications; however, siRNAs are not readily bioavailable. Cationic liposomes offer effective protection of drug from nucleases and enable distribution to desired target organs. The amount of siRNA in the formulation must be accurately determined. We have developed a stability-indicating ion-pair reversed-phase high-performance liquid chromatography method to separate and accurately quantitate two siRNA duplexes in a liposome without sample pretreatment. The gradient mobile phase system consisted of 385 mM hexafluoro-2-propanol, 14.5 mM triethylamine, and 5% methanol and 385 mM hexafluor... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3284589 Fri, 12 Feb 2010 00:00:00 +0100 3284589 http://www.medworm.com/index.php?rid=3284588&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20156416%26dopt%3DAbstract Authors: Wentzel JF, Gouws C, Huysamen C, Dyk EV, Koekemoer G, Pretorius PJ The comet assay (single cell gel electrophoresis) is a cost-effective, sensitive and simple technique which is traditionally used for analysing and quantifying DNA damage in individual cells. The aim of this study was to determine whether the comet assay could be modified to detect changes in the levels of DNA methylation in single cells. We used the difference in methylation sensitivity of the isoschizomeric restriction endonucleases Hpall and Mspl to demonstrate the feasibility of the comet assay to measure the global DNA methylation level of individual cells. The results were verified with the well established cytosine extension assay. We were able to show variations in DNA methylation after treatment of cul... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3284588 Fri, 12 Feb 2010 00:00:00 +0100 3284588 http://www.medworm.com/index.php?rid=3284587&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20156417%26dopt%3DAbstract Authors: Stefanowicz P, Kijewska M, Kluczyk A, Szewczuk Z The products of non-enzymatic glycation of proteins are formed in chemical reaction between reducing sugars and the free amino group located either at the N-terminus of the polypeptide chain or in the lysine side chain. The glycated proteins as well as their fragments could be used as markers of the aging process as well as diabetes mellitus and Alzheimer's disease, which makes them an object of the interest in clinical chemistry. In this paper we propose a new method for the identification of peptide - derived Amadori products in the mixtures obtained by enzymatic hydrolysis of glycated proteins. Two proteins: ubiquitin and human serum albumin (HSA) were modified with the equimolar mixture of glucose and [(13)C(6)]glucose and s... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3284587 Fri, 12 Feb 2010 00:00:00 +0100 3284587 http://www.medworm.com/index.php?rid=3284586&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20156418%26dopt%3DAbstract Authors: Landino LM, Brown CM, Edson CA, Gilbert LJ, Grega-Larson N, Wirth AJ, Lane KC Numerous studies of S-glutathionylation of cysteine thiols indicate that this protein modification plays a key role in redox regulation of proteins. To facilitate the study of protein S-glutathionylation, we developed a synthesis and purification to produce milligram quantities of fluorescein-labeled glutathione. The amino terminus of the glutathione tripeptide reacted with fluorescein isothiocyanate readily in ammonium bicarbonate. Purification by solid phase extraction on C8 and C18 columns separated excess reactants from desired products. Both oxidized and reduced fluorescein-labeled glutathione reacted with a variety of thiol containing proteins to yield fluorescent proteins. PMID: 20156418 [... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3284586 Fri, 12 Feb 2010 00:00:00 +0100 3284586 http://www.medworm.com/index.php?rid=3276791&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20153711%26dopt%3DAbstract Authors: Liou YB, Sheu MT, Liu DZ, Lin SY, Ho HO A sensitive and accurate normal-phase liquid chromatographic and atmospheric pressure chemical ionization mass spectrometric (normal-phase LC-APCI-MS) method for determining the standard ceramide [NS] (Cer[NS]) was developed and validated in order to improve on the traditional thin-layer chromatographic (TLC) technique and LC-electrospray ionization (ESI)-MS method to profile and quantify ceramides in nude mouse skin. Normal-phase LC-APCI-MS was optimized to separate the nine classes of ceramides presented in the stratum corneum (SC) of nude mouse skin. A normal-phase silica column eluted with the gradient system from heptane: acetone/butanol (90:10, v/v) of 75%: 25% to 100% acetone/butanol (90:10, v/v) (with each solvent containing 0.1%... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3276791 Thu, 11 Feb 2010 00:00:00 +0100 3276791 http://www.medworm.com/index.php?rid=3276792&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20153286%26dopt%3DAbstract This study demonstrates that some of the commonly used reference genes are inadequate for normalization of Q-PCR data, due to their expression instability. Further, this study validates HPRT1 and RPLP0 as appropriate reference genes for Q-PCR analysis in the CIH model. PMID: 20153286 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3276792 Wed, 10 Feb 2010 00:00:00 +0100 3276792 http://www.medworm.com/index.php?rid=3276796&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20152791%26dopt%3DAbstract We present a new application of the non-competitive phage anti immunocomplex assay (PHAIA) by converting an existing competitive assay to a versatile non-competitive sandwich type format utilizing an immunocomplex binding phage-borne peptides to detect the brominated flame retardant, BDE 47. Three phage-displayed 9-mer disulfide constrained peptides were isolated that recognize the BDE 47-polyclonal antibody immunocomplex. The resulting PHAIAs showed variable sensitivities and the most sensitive peptide had a dose-response curve with the SC(50) of 0.7 ng/ml of BDE 47 and linear range of 0.3 - 2 ng/ml which was nearly identical to the best heterologous competitive format (IC(50) of 1.8 ng/ml, linear range of 0.4-8.5/ml). However, the PHAIA was 1400-fold better than homologous competitive as... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3276796 Tue, 09 Feb 2010 00:00:00 +0100 3276796 http://www.medworm.com/index.php?rid=3276795&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20152792%26dopt%3DAbstract Authors: Xu Y, Wang J, Bao Y, Jiang W, Zuo L, Song D, Hong B, Si S CD36, a class B scavenger receptor, is an integral membrane protein that mediates the endocytosis of modified lipoproteins. The functions of CD36 are complex and have been associated with atherosclerosis. In the present study, we developed a high-throughput screening (HTS) assay to identify small molecule antagonists by expressing human CD36 using a Bac-to-Bac baculovirus expression system in Spodoptera frugiperda (Sf9) cells. Uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled Ac-LDL (DiI-AcLDL) revealed that the IC(50) values for the CD36 ligands Ox-LDL, Ac-LDL and HDL were 0.039, 0.019 and 0.010 mug/ml, respectively. Using the HTS assay, two novel compounds, 2016481B and 2038751B, wer... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3276795 Tue, 09 Feb 2010 00:00:00 +0100 3276795 http://www.medworm.com/index.php?rid=3276794&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20152793%26dopt%3DAbstract The objective of this study is to empirically validate all of the performance characteristics predicted by Ekins' theory. Flow cytometric analysis was used to detect binding between a fluorescent ligand and capture microparticles since it can directly measure fractional occupancy, the primary response variable in ambient analyte theory. After experimentally determining ambient analyte conditions, comparisons were carried out between ambient and non-ambient assays in terms of their signal strengths, limits of detection, and their sensitivity to variations in reaction volume and number of particles. The critical number of binding sites required for an assay to be in the ambient analyte region was estimated to be 0.1VKd. As predicted, such assays exhibited superior signal/noise levels and lim... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3276794 Tue, 09 Feb 2010 00:00:00 +0100 3276794 http://www.medworm.com/index.php?rid=3276793&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20152794%26dopt%3DAbstract Authors: Liu H, Chumsae C, Gaza-Bulseco G, Goedken ER Human IgG1 contains twelve domains and each has an intra chain disulfide bond that connects the two layers of anti-parallel beta-sheets. These intra chain disulfide bonds are shielded from solvents under native conditions. Therefore, accessibility of the disulfide bonds to reduction under conditions that unfold antibody has the potential to be a good indicator of the thermodynamic stability of each domain. The stability of a recombinant monoclonal antibody at the domain level was investigated using a novel method involving reduction of the disulfide bonds in the presence of increasing amounts of guanidine hydrochloride and alkylation with (12)C-iodoacetic acid, which was followed by reduction of the remaining disulfide bonds and alk... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3276793 Tue, 09 Feb 2010 00:00:00 +0100 3276793 http://www.medworm.com/index.php?rid=3268579&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20149779%26dopt%3DAbstract Authors: Park EJ, Na DH Microchip electrophoretic method was applied to monitor and characterize the PEGylation of the proteins, alpha-lactalbumin and bovine serum albumin, using several poly(ethylene glycol) (PEG) derivatives with molecular weights from 1 to 20 kDa. This method effectively separated multi-PEGylated proteins in a size-based manner and allowed monitoring of the PEGylation pattern with the advantages of high speed, minimal sample consumption, and high reproducibility. Microchip electrophoresis would be a very useful tool for protein PEGylation studies, such as reaction monitoring, purity checks and characterization of PEGylated protein products. PMID: 20149779 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3268579 Mon, 08 Feb 2010 00:00:00 +0100 3268579 http://www.medworm.com/index.php?rid=3268578&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20149780%26dopt%3DAbstract Authors: Giehm L, Otzen DE There is great interest in developing reproducible high-throughput screens to identify small molecular inhibitors of protein fibrillization and aggregation for possible therapy against deposition diseases such as Alzheimer's and Parkinson's (PD). We have made a methodical analysis of factors increasing the reproducibility of the fibrillization of alpha-synuclein (alphaSN), a 140 amino acid protein implicated in PD and notorious for its erratic fibrillization behaviour. Salts and polyanionic polymers do not significantly improve the quality of the assay. However, an orbital agitation mode in the plate reader is a crucial first step towards reproducible alphaSN fibrillization. Higher reproducibility is achieved by the addition of glass beads, as evaluated by th... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3268578 Mon, 08 Feb 2010 00:00:00 +0100 3268578 http://www.medworm.com/index.php?rid=3244651&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20123080%26dopt%3DAbstract Authors: Wisnewski AV, Liu J, Redlich CA Diphenylmethane diisocyanate (MDI), the chemical commonly used as a cross-linking agent in commercial polyurethane production, is a well-recognized cause of asthma. Reaction products between MDI and "self" proteins are hypothesized to act as antigens capable of inducing airway inflammation and asthma; however, such MDI antigens remain incompletely understood. We utilized a variety of analytical methods to characterize the range of MDI-albumin reaction products that form under physiologic conditions. Sites of MDI conjugation on antigenic MDI-albumin products, as defined by serum IgG from MDI exposed workers, were determined by high performance liquid chromatography (HPLC), followed by tandem mass spectrometry (MS/MS). The data identified fourteen... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3244651 Sat, 30 Jan 2010 00:00:00 +0100 3244651 http://www.medworm.com/index.php?rid=3244650&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20123081%26dopt%3DAbstract Authors: Zhou S, Lu X, Chen C, Sun D A new immunoassay method called Specific Analyte Labeling and Recapture Assay (SALRA) to quantitatively measure protein abundance was developed and the assay conditions were optimized. The key features of this method include labeling the antigen bound to the capture antibody, eluting the labeled antigen and recapturing it by the same capture antibody on the detection plate. The reporter molecules on the labeled antigen provide a convenient and reliable means for signal detection. We demonstrated that the dose-response curve of SALRA was comparable to that of sandwich enzyme-linked immunosorbent assays (ELISA), and better than that of the antigen direct-labeling method. In addition, multiple proteins can be simultaneously measured by SALRA. Using the... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3244650 Sat, 30 Jan 2010 00:00:00 +0100 3244650 http://www.medworm.com/index.php?rid=3244649&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20123082%26dopt%3DAbstract Authors: Martínez-Ceron MC, Giudicessi SL, Marani MM, Albericio F, Cascone O, Erra-Balsells R, Camperi SA Optimisation of beads analysis by MALDI-TOF-MS after the screening of one-bead-one-peptide combinatorial libraries was achieved, involving the fine tuning of the whole process. Guanidine was replaced by MeCN/AcOH/H(2)O, this improving matrix crystallisation. Peptide-bead cleavage with NH(4)OH was cheaper, safer and as efficient as NH(3)/THF. Peptide elution in micro tubes instead of placing the beads in the sample plate yielded more sample aliquots. "Successive-dry-layers-deposit" sample preparation was better than "dried-droplet method". Among the matrices analysed, alpha-cyano-4-hydroxycinnamic acid resulted in the best peptide ion yield. Cluster formation was minimised by a... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3244649 Sat, 30 Jan 2010 00:00:00 +0100 3244649 http://www.medworm.com/index.php?rid=3244648&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20123083%26dopt%3DAbstract In this study, a magnetic-bead-based platform amenable to high-throughput protein carbonic anhydrase II (CA II) capture is presented. The key steps in this approach involved immunoaffinity purification of the target protein from serum followed by on-bead digestion with trypsin to release a surrogate peptide. This tryptic peptide was quantified by liquid chromatography/electrospray-tandem mass spectrometry (LC/ESI-MS/MS) operating in Multi Reaction Monitoring acquisition mode. Using a synthetic peptide standard and a structural analogue free-labeled internal standard, the resulting concentration was stoichiometrically converted to CA II serum concentration. The analytical steps, such as preparation of immunobeads, protein capture, proteolysis and calibration were optimized. The method was v... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3244648 Sat, 30 Jan 2010 00:00:00 +0100 3244648 http://www.medworm.com/index.php?rid=3244656&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20122888%26dopt%3DAbstract Authors: Wakiyama M, Kaitsu Y, Matsumoto T, Yokoyama S Cell-free protein synthesis is one of the best methods to express recombinant proteins efficiently. We developed a transcription and translation coupled (TnT) cell-free system from Drosophila S2 cells. To improve the translational efficiency of mRNA lacking a 5'-cap structure, we employed the sequence of the 5'-untranslated region derived from baculovirus p10 mRNA. By using this sequence, we established a PCR fragment-based TnT system. FLAG-tagged human Argonaute2 and TRBP2 were successfully synthesized, within 90 minutes in a reaction volume of less than 10 mul, and were detected by the FLAG antibody. PMID: 20122888 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3244656 Fri, 29 Jan 2010 00:00:00 +0100 3244656 http://www.medworm.com/index.php?rid=3244655&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20122889%26dopt%3DAbstract Authors: Masood MA, Yuan C, Acharya JK, Veenstra TD, Blonder J Sphingomyelin (SM) and ceramide-phosphoethanolamines (cer-PE) are related lipids present in mammals and insects, respectively. Owing to the critical roles that cer-PE play in eukaryotic cellular function, there is a need to develop methods that provide accurate quantitation of these compounds. Results obtained in this study demonstrate that Drosophila contains cer-PE's with unsaturated sphingoid base cores as well as low levels of cer-PE's that possess saturated sphingoid base cores. Specifically, the method developed in this study enabled the quantitation of picogram amounts of cer-PE containing both unsaturated d14:1(Delta4) and d16:1(Delta4) and saturated d14:0 sphingoid base cores. Using this method cer-PE compounds wit... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3244655 Fri, 29 Jan 2010 00:00:00 +0100 3244655 http://www.medworm.com/index.php?rid=3244654&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20122890%26dopt%3DAbstract Authors: Saeki S, Kunitomo H, Narita Y, Mimura H, Nishi T, Sasaki K We have established a cAMP response element (CRE)-mediated reporter assay system for G-protein-coupled receptors (GPCRs) using an oriP-based estrogen-inducible expression vector and the B-cell line (GBC53 or GBCC71) that expresses EBNA-1 and is adapted to serum-free culture. GBC53 harbors a GAL4-ER expression unit and a CRE-luciferase gene in the genome, and GBCC71 additionally harbors expression units for two chimeric Galphas proteins (Gs/q and Gs/i). Introduction of a GPCR-expression plasmid into GBC53 or GBCC71 creates polyclonal stable transformants in two weeks, which are easily expanded and used for assays after induction of the GPCR expression. Using GBC53, we detected ligand-dependent signals of Gs-coupled GPCR... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3244654 Fri, 29 Jan 2010 00:00:00 +0100 3244654 http://www.medworm.com/index.php?rid=3244653&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20122891%26dopt%3DAbstract Authors: Bravo SB, Garcia-Rendueles ME, Perez-Romero S, Cameselle-Teijeiro J, Rodrigues JS, Barreiro F, Alvarez CV Recently, it was shown that commercial human thyroid lines were in fact derived from colon, mammary carcinoma or melanoma. Others demonstrated the absence of a common pattern of gene expression between available thyroid-cancer cell lines and tumours from patients. Thus, it is important to use several primary cells with a common pathological origin to achieve reproducible results, and necessary to find common methods for manipulation of protein expression in such various cultures. We standardized a transfection method for efficient expression of exogenous proteins in human primary thyroid cultures. We compared lipid-based techniques with three electroporation systems (Pulse... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3244653 Fri, 29 Jan 2010 00:00:00 +0100 3244653 http://www.medworm.com/index.php?rid=3244652&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20122892%26dopt%3DAbstract Authors: Mohanty JG, Bhamidipaty S, Evans MK, Rifkind JM Oxidative stress, originating from reactive oxygen species (ROS), has been implicated in aging and various human diseases. The ROS generated can oxidize proteins producing protein carbonyl derivatives. The level of protein carbonyls in blood plasma has been used as a measure of overall oxidative stress in the body. Classically, protein carbonyls have been quantitated spectrophotometrically by directly reacting them with 2,4, dinitrophenylhydrazine (DNPH). However, the applicability of this method to biological samples is limited by its low inherent sensitivity. This limitation has been overcome by the development of sensitive ELISA methods to measure protein carbonyls. As part of the Healthy Aging in Neighborhoods of Diversity ac... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3244652 Fri, 29 Jan 2010 00:00:00 +0100 3244652 http://www.medworm.com/index.php?rid=3236687&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20117070%26dopt%3DAbstract Authors: Teixeira LD, Grasso AN, Monteiro AM, Figueiredo Neto AM, Vieira Jr ND, Gidlund M, Courrol LC Low-density lipoprotein (LDL) particles are the major cholesterol-carrying lipoprotein in the human circulation from the liver to peripheral tissues. High levels of LDL cholesterol (LDL-C) are known risk factor for the development of coronary artery disease (CAD). The most common approach to determine the LDL-C in the clinical laboratory involves the Friedewald formula. However, in certain situations, this approach is inadequate. In this paper we report on the enhancement on the Europium emission band of Europium chlorotetracycline complex (CTEu) in the presence of LDL. The emission intensity at 615 nm of the CTEu increases with increasing amounts of LDL. This phenomenon allowed us to ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3236687 Thu, 28 Jan 2010 00:00:00 +0100 3236687 http://www.medworm.com/index.php?rid=3236686&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20117071%26dopt%3DAbstract Authors: Yen TY, Stephen Inbaraj B, Chien JT, Chen BH A gas chromatography-mass spectrometry (GC-MS) method was developed to simultaneously separate cholesterol, 8 cholesterol oxidation products (COPs) and 2 conjugated linoleic acids (9-cis, 11-trans CLA, 10-trans, 12-cis CLA), and evaluate their stability in a model system during heating. Among 4 capillary columns tested, an Equity-5 column with low-polar stationary phase provided better resolution within 30 min. An HPLC method was also developed to determine cholesterol hydroperoxides by using YMC C30 column with diphenyl-1-pyrenylphosphine as fluorescence reagent. No formation of COPs and degradation of cholesterol and CLAs occurred at 100 degrees C, but the levels of COPs rose drastically at 150 degrees C. The first-order rate of c... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3236686 Thu, 28 Jan 2010 00:00:00 +0100 3236686 http://www.medworm.com/index.php?rid=3223896&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20109435%26dopt%3DAbstract Authors: Adhikari S, Manthena PV, Sajwan K, Kota KK, Roy R Protein purification is still very empirical, and a unified method to purify proteins without an affinity tag is not available yet. In the post-genomic era, functional genomics, however, strongly demands such a method. In this paper we have formulated a unique method that can be applied to purify any recombinant basic protein from E. coli. Here, we have found that if the pH of the buffer is merely one pH unit below the isoelectric point (pI) of the recombinant proteins, most of the latter bind to the column. This result supports the Henderson-Hasselbalch principle. Considering that E. coli proteins are mostly acidic, and based on the pI determined theoretically, apparently all recombinant basic proteins (at least pI-16.94) may ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3223896 Mon, 25 Jan 2010 00:00:00 +0100 3223896 http://www.medworm.com/index.php?rid=3223895&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20109436%26dopt%3DAbstract Authors: Jones B, Holskin B, Meyer S, Ung T, Dupriez V, Flores SY, Burgeon E, Ator M, Duzic E Assay technologies that measure intracellular Ca(2+) release are among the predominant methods for evaluation of GPCR function. These measurements have historically been performed using cell-permeable fluorescent dyes, although use of the recombinant photoprotein aequorin (AEQ) as a Ca(2+) sensor has gained popularity with recent advances in instrumentation. The requirement of the AEQ system for cells expressing both the photoprotein as well as the GPCR target of interest has necessitated the labor-intensive development of cell lines stably expressing both proteins.With the goal of streamlining this process, transient transfections were used to either 1) introduce AEQ into cells stably express... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3223895 Mon, 25 Jan 2010 00:00:00 +0100 3223895 http://www.medworm.com/index.php?rid=3223894&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20109437%26dopt%3DAbstract Authors: Triguero A, Cabrera G, Royle L, Harvey DJ, Rudd PM, Dwek RA, Bardor M, Lerouge P, Cremata JA Plants synthesize N-glycans containing the antigenic sugars alpha(1,3)-fucose and beta(1,2)-xylose. Therefore it is important to monitor these N-glycans in monoclonal antibodies produced in plants (plantibodies). We evaluate several techniques to characterize the N-glycosylation of a plantibody produced in tobacco plants with and without the KDEL tetrapeptide endoplasmic reticulum retention signal which should inhibit or drastically reduce the addition of alpha(1,3)-fucose and beta(1,2)-xylose. Ammonium hydroxide/carbonate based chemical deglycosylation and PNGase A enzymatic releasing were investigated giving similar 2-aminobenzamide labeled N-glycans HPLC profiles. The chemical relea... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3223894 Mon, 25 Jan 2010 00:00:00 +0100 3223894 http://www.medworm.com/index.php?rid=3212848&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20100450%26dopt%3DAbstract We present an on-line LC/MS method that uses high-low cone voltage switching for in-source fragmentation to determine the structures of the complex glycans present on each site for the two gene products of AAG. High cone voltage caused carbohydrate fragmentation leading to the generation of signature carbohydrate ions that we used as markers to identify the glycopeptides. Low cone voltage produced minimal carbohydrate fragmentation and enabled the identification and quantification of the intact oligosaccharide structures on each glycopeptide based on its monoisotopic mass and intensity. Quantitation was accomplished by using the intensities of peaks from deconvoluted and de-isotoped mass spectra or from the areas of the extracted ion chromatograms from the tryptic peptide maps. The combine... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3212848 Fri, 22 Jan 2010 00:00:00 +0100 3212848 http://www.medworm.com/index.php?rid=3212847&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20100451%26dopt%3DAbstract Authors: Poon GM Isothermal titration calorimetry (ITC) produces a differential heat signal with respect to the total titrant concentration. This feature gives ITC excellent sensitivity for studying the thermodynamics of complex biomolecular interactions in solution. Currently, numerical methods for data fitting are based primarily on indirect approaches rooted in the usual practice of formulating biochemical models in terms of integrated variables. Here, a direct approach is presented wherein ITC models are formulated and solved as numerical initial value problems for data fitting and simulation purposes. To do so, the ITC signal is cast explicitly as a first-order ordinary differential equation (ODE) with total titrant concentration as independent variable and the concentration of a ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3212847 Fri, 22 Jan 2010 00:00:00 +0100 3212847 http://www.medworm.com/index.php?rid=3212846&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20100452%26dopt%3DAbstract Authors: Arezi B, McCarthy M, Hogrefe H Inhibitor-resistance of several commercial MMLV RT enzymes was investigated. IC(50) values were determined for potential RNA contaminants, including guanidine thiocyanate, ethanol, formamide, EDTA, and plant-related acidic polysaccharides. Sensitivity (as judged by MMLV RT IC(50) values) was directly correlated to the outcome of "mock" RT-qPCR assays carried out with exogenous inhibitors. MMLV RT enzymes lacking RNase H activity were shown to be more sensitive to RT-qPCR inhibitors. In contrast, a thermal-resistant MMLV RT pentuple mutant (E69K/E302R/W313F/L435G/N454K) showed higher tolerance to these substances than wild-type. Increased resistance was also noted in RT-qPCR comparisons employing crude cell lysates. PMID: 20100452 [PubMed - as... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3212846 Fri, 22 Jan 2010 00:00:00 +0100 3212846 http://www.medworm.com/index.php?rid=3212850&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20096259%26dopt%3DAbstract Authors: Gussoni M, Cremonini MA, Vezzoli A, Greco F, Zetta L A NMR method was implemented to assess 'in vivo' oxygenation levels by a quantitative determination of the (1)H-NMR resonances of Mb: the proximal His-F8 N(delta)H at 70-90 ppm and Val-E11 gammaCH(3) resonance at -2.8 ppm, reflecting deoxygenated (deoxy-Mb) and oxygenated (met-Mb) states, were alternately recorded. The method was developed 'in vitro' choosing a couple of NMR sequences that could each maximize the SNR, while avoiding baseline rolling and suppressing the water signal. Two quantitative calibration methods were implemented for deoxy- and met-Mb samples (0.1 -1mM), respectively. LOD and LOQ resulted 0.015 mM and 0.05 mM for MetMb and 0.013 mM and 0.042 mM for deoxy-Mb. Sequences and calibration curves were employ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3212850 Tue, 19 Jan 2010 00:00:00 +0100 3212850 http://www.medworm.com/index.php?rid=3212849&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20096260%26dopt%3DAbstract Authors: Ahmad S, Carter JJ, Scott JE PON1 is a high density lipoprotein-associated enzyme that plays an important role in organophosphate detoxification and prevention of atherosclerosis. Thus, there is significant interest in identifying nutritional and pharmacological enhancers of PON1 activity. In order to identify such compounds, we developed a rapid homogeneous assay to detect endogenous cell-associated PON1 activity. PON1 activity was measured by the simple addition of fluorigenic PON1 substrate DEPFMU to live Huh7 cells in media and monitoring change in fluorescence. A specific PON1 inhibitor, 2-hydroxyquinoline, was used to confirm that the observed activity was due to PON1. The assay was optimized and characterized with regard to time course, substrate and sodium chloride con... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3212849 Tue, 19 Jan 2010 00:00:00 +0100 3212849 http://www.medworm.com/index.php?rid=3200972&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20093103%26dopt%3DAbstract In this study, we aimed to isolate an RNA aptamer to Cy3, a widely-used and membrane-permeant and non-toxic fluorescent cyanine dye. Extensive selection of affinity RNA molecules to Cy3 yielded a unique sequence aptamer named Cy3_apt. The selected Cy3_apt was 83 nucleotides long and successfully shortened to 49 nucleotides long with increased affinity to Cy3 by multiple base changes. The shortest Cy3_apt is composed of two separate hairpin modules that are required for the affinity to Cy3 as monitored by the surface plasmon resonance (SPR) assay. Also, the fluorescence of Cy3 increased upon binding to Cy3_apt. The two modules of Cy3_apt, when detached from each other functioned as a binary aptamer probe. We demonstrate that the binary Cy3_apt probe is applicable to the detection of target ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3200972 Mon, 18 Jan 2010 00:00:00 +0100 3200972 http://www.medworm.com/index.php?rid=3194572&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20085742%26dopt%3DAbstract Authors: Gu S, Wen D, Weinreb PH, Sun Y, Zhang L, Foley SF, Kshirsagar R, Evans D, Mi S, Meier W, Pepinsky Blake R Trisulfides are a post-translational modification formed by the insertion of a sulfur atom into a disulfide bond. Although reports for trisulfides in proteins are limited, we find that they are a common modification in natural and recombinant antibodies of all IgG subtypes. Trisulfides were only detected in interchain linkages and were predominantly in the light-heavy linkages. Factors that lead to trisulfide formation and elimination and their impact on activity and stability were investigated. The peptide mapping methods developed for characterization and quantification of trisulfide should be applicable to any antibody and can be easily adapted for other types of protei... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3194572 Sat, 16 Jan 2010 00:00:00 +0100 3194572 http://www.medworm.com/index.php?rid=3194571&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20085743%26dopt%3DAbstract We describe a new low-ionic-strength sodium threonine (STh) medium with the advantage of avoiding relative DNA band migration changes following electrophoresis of supercoiled DNA in agarose gel, when substituted for the standard conductive media of Tris-boric acid-ethylenediaminetetraacetic acid (EDTA) (TBE), Tris-acetic acid-EDTA (TAE) and the low-ionic-strength medium, sodium boric acid (SB). Low-ionic-strength STh medium provided better resolution, less heat generation and prevention of relative migration order changes among linear, covalently closed circular- (ccc) and open circular- (oc) formed DNA in the range of 2-10 kbp in 1% agarose gel electrophoresis. PMID: 20085743 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3194571 Sat, 16 Jan 2010 00:00:00 +0100 3194571 http://www.medworm.com/index.php?rid=3194570&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20085744%26dopt%3DAbstract Authors: Oshige M, Yamaguchi K, Matsuura SI, Kurita H, Mizuno A, Katsura S A simple molecular combing method for analysis of biochemical reactions, called the moving droplet method, has been developed. In this method, small droplets containing DNA molecules run down a sloped glass substrate, and this creates a moving interface between the air, droplet, and substrate, which stretches the DNA molecules. This method requires a much smaller volume of sample solution than other established combing methods, and this allows for wider application in various fields. Using this method, lambdaDNA molecules were stretched and absorbed to a glass substrate, and single-molecule analysis of DNA synthesis by DNA polymerases was performed. PMID: 20085744 [PubMed - as supplied by publisher] (Source:... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3194570 Sat, 16 Jan 2010 00:00:00 +0100 3194570 http://www.medworm.com/index.php?rid=3194575&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20083082%26dopt%3DAbstract Authors: Chung MK, Riby J, Li H, Iavarone AT, Williams ER, Zheng Y, Rappaport SM Adducts of benzo[a]pyrene-diolepoxide (BPDE) with blood nucleophiles have been used as biomarkers of exposure to polycyclic aromatic hydrocarbons (PAHs). The most popular such assay is a competitive ELISA which employs monoclonal antibody 8E11 to detect benzo[a]pyrene tetrols following hydrolysis of BPDE adducts from lymphocyte DNA or human serum albumin (HSA). Here we use 8E11 as the capture antibody in a sandwich ELISA to detect BPDE-HSA adducts directly in 1 mg samples of HSA or 20 muL of serum/plasma. The assay employs an anti-HSA antibody for detection, which is amplified by an avidin/biotinylated horseradish peroxidase complex. The sandwich ELISA has advantages of specificity and simplicity and is ab... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3194575 Fri, 15 Jan 2010 00:00:00 +0100 3194575 http://www.medworm.com/index.php?rid=3194574&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20083083%26dopt%3DAbstract Authors: Wang L, Li PC A microfluidic microarray device, which has been developed for parallel DNA detection, is now further optimized for more rapid and sensitive DNA detection, and for the single-base-pair discrimination of two fungal pathogenic PCR products. Two PDMS-based microfluidic chips consist of radial and spiral microchannels in which flexible probe creation and convenient sample delivery have been achieved by centrifugal pumping. The microarray hybridizations occurred at the cross sections within the spiral channels intersecting the pre-printed radial probe lines. The centrifugal pumping method showed advantages over the vacuum suction method in terms of parallel solution delivery and less signal variations between duplicated samples. The effect of microchannel depth was st... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3194574 Fri, 15 Jan 2010 00:00:00 +0100 3194574 http://www.medworm.com/index.php?rid=3194573&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20083084%26dopt%3DAbstract Authors: Rozum B, Gajownik K, Tymecki L, Koncki R One-step carbodiimide method was found to allow covalent binding of enzymes to the inner wall of PVC tubing. The immobilization is performed under mild conditions without neither laborious pretreatment nor activation of reactor surface. In these preliminary studies alkaline phosphatase (ALP, EC 3.1.3.1) and p-nitrophenyl phosphate (NPP) were applied as a model enzyme and substrate, respectively. The resulting open-tubular bioreactor exhibits satisfactory operational and storage stability. Additionally, a novel, very simple instrumental concept for optical monitoring of biocatalytic process directly inside the microbioreactor using a system of paired emitter-detector diodes is presented. PMID: 20083084 [PubMed - as supplied by publis... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3194573 Fri, 15 Jan 2010 00:00:00 +0100 3194573 http://www.medworm.com/index.php?rid=3186252&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20079705%26dopt%3DAbstract Authors: Khoury GE, Laurenceau E, Chevolot Y, Mérieux Y, Desbos A, Fabien N, Rigal D, Souteyrand E, Cloarec JP Protein microarray technology provides a useful approach for the simultaneous serodetection of various antibodies in low sample volumes. In order to implement functional protein microarrays, appropriate surface chemistry has to be designed so that both the protein structure and the biological activity can be retained. In the present study, two surface chemistries for protein microarrays and immunofluorescent assays have been developed. Glass slides were functionalized with NHS-ester via a monofunctional silane or Maleic Anhydride-alt-Methyl Vinyl Ether (MAMVE) copolymer to allow covalent grafting of histone proteins. Analytical performance of these microarrays were then e... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3186252 Wed, 13 Jan 2010 00:00:00 +0100 3186252 http://www.medworm.com/index.php?rid=3186251&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20079706%26dopt%3DAbstract Authors: Okello JB, Zurek J, Devault AM, Kuch M, Okwi AL, Sew.ambo NK, Bimenya GS, Poinar D, Poinar HN Archival formalin-fixed paraffin-embedded (FFPE) human tissue collections are typically in poor states of storage across the developing world. With advances in bio-molecular techniques, these extraordinary and virtually untapped resources have become an essential part of retrospective epidemiological studies. To successfully utilize such tissues in genomic studies, scientists require high nucleic acid yields and purity. In spite of the increasing number of FFPE tissues kits available, few studies have analyzed their applicability in recovering high-quality nucleic acids from archived human autopsy samples. Here, we provide a study involving 10 major extraction methods used to isolate ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3186251 Wed, 13 Jan 2010 00:00:00 +0100 3186251 http://www.medworm.com/index.php?rid=3177893&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20074539%26dopt%3DAbstract Authors: Ye H, Hill J, Kauffman J, Han X The capability of iTRAQ (isotope tags for relative and absolute quantification) reagents coupled with MALDI-TOF/TOF-MS as a qualitative and quantitative technique for the analysis of complicated protein pharmaceutical mixtures was evaluated. Mixtures of Somavert and Miacalcin with a small amount of BSA (bovine serum albumin) as an impurity were analyzed. Both Somavert and Miacalcin were qualitatively identified, and BSA was detected at levels as low as 0.8 mol%. Genotropin and Somavert were compared in a single experiment, and all the distinct amino acid residues from the two proteins were readily identified. Four somatropin drug products (Genotropin, Norditropin, Jintropin, and Omnitrope) were compared using the iTRAQ/MALDI-MS method to determi... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3177893 Mon, 11 Jan 2010 00:00:00 +0100 3177893 http://www.medworm.com/index.php?rid=3177892&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20074540%26dopt%3DAbstract Authors: Magdalena W, Adam L, Grażyna M, Anna L, Krzysztof R Previously selected by the combinatorial chemistry approach potent fluorogenic substrate of proteinase 3 was used as the starting structure to design new substrates. The general formula of the synthesized peptides is as follows: ABZ-Tyr-Tyr-Abu-ANB-X-NH(2), where ANB (stands for 5-amino-2-nitrobenzoic acid) served as a chromofore and an acceptor of fluorescence, ABZ (aminobenzoic acid) is a donor of fluorescence in these FRET peptides and X is proteinogenic amino acid (except Cys). The introduced modifications influenced substrate activity of the synthesized peptides. The highest value of specificity constant for proteinase 3 was obtained for the single peptide with Gln in the discussed position (k(cat)/K(M) = 275,000 M... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3177892 Mon, 11 Jan 2010 00:00:00 +0100 3177892 http://www.medworm.com/index.php?rid=3177885&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20074541%26dopt%3DAbstract We report here the details of this method and its use for identification of 10 plasma and urinary metabolites in treated subjects, including indole species in urine that are not themselves metabolites of SPB. This approach thus contributes to understanding metabolic pathways that differ among HD individuals being treated with SPB. PMID: 20074541 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3177885 Mon, 11 Jan 2010 00:00:00 +0100 3177885 http://www.medworm.com/index.php?rid=3177884&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20074542%26dopt%3DAbstract Authors: Kotia RB, Raghani AR Signal peptides used in biosynthesis of proteins are cleaved at a very specific site by signal peptidase during post-translational translocation of cytoplasmic proteins across the membrane. In some cases though, there can be cleavage at non-specific sites, giving rise to heterogeneity in the mature protein, which manifests itself as either elongation or truncation of the N-terminus of the mature protein. When used as biopharmaceutical therapeutics, such heterogeneities may be a cause of concern, depending on the nature of the heterogeneity. This paper describes the determination of such heterogeneity by peptide mapping in both the heavy chain and the light chain of a Chinese Hamster Ovary (CHO) cell-expressed monoclonal antibody. The peptide map method des... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3177884 Mon, 11 Jan 2010 00:00:00 +0100 3177884 http://www.medworm.com/index.php?rid=3177883&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20074543%26dopt%3DAbstract We report a method to enrich cysteinyl adducts of human serum albumin (HSA)(2), representing biomarkers of exposure to systemic electrophiles. Because the major site of HSA adduction is the single free sulfhydryl group at Cys(34), we used thiol-affinity resins to remove mercaptalbumin (i.e., unadducted HSA) from the cysteinyl adducts. Electrospray ionization mass spectrometry was used to detect mercaptalbumin and HSA-Cys(34) modifications before and after enrichment of HSA. Differences in adduct content were detected across samples of freshly-isolated, archived, and commercial HSA. Cysteinylated and glycosylated adducts were present in all samples with abundances decreasing in the order: commercial HSA > archived HSA > fresh HSA. After enrichment of HSA, mercaptalbumin was no longer ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3177883 Mon, 11 Jan 2010 00:00:00 +0100 3177883 http://www.medworm.com/index.php?rid=3177882&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20074544%26dopt%3DAbstract Authors: Peng L, Kinsel GR Previous studies in our group have shown that the analyte signal in a MALDI MS experiment is strongly influenced by the binding interactions between the target surface and the analyte. Specifically, the analyte signal increases with decreases in surface binding affinity, which has been attributed to more unbound analyte being available for incorporation within the MALDI matrix. In this work polyethylene glycol (PEG) has been chemically grafted onto a polyurethane (PU) film to produce a MALDI target having reduced surface-protein binding affinity and the effect of this modification on protein MALDI ion signals has been investigated. The proteins myoglobin, lysozyme and albumin have been used to evaluate the PEG PU modified target as compared with a PU target a... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3177882 Mon, 11 Jan 2010 00:00:00 +0100 3177882 http://www.medworm.com/index.php?rid=3177881&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20074545%26dopt%3DAbstract Authors: Martin-Harris MH, Bartley PA, Morley AA We developed a simple and robust method for removing non-specific amplification produced during gene walking with a gene-specific and a degenerate primer. The primary walking PCR was followed by 2-3 PCR rounds, each incorporating a low concentration of a reverse hybrid primer, of which the 3' end bound to a target sequence generated in the preceding PCR round and the 5' end was a new sequence which generated a target sequence for the next PCR round. The low concentration of the hybrid primer and the extent of amplicon stem-loop formation inhibited non-specific amplification and enabled successful walking along 3 genes. PMID: 20074545 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3177881 Mon, 11 Jan 2010 00:00:00 +0100 3177881 http://www.medworm.com/index.php?rid=3172317&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20064482%26dopt%3DAbstract Authors: Matsumoto F, Deshimaru S, Oda T, Fujiwara S We have developed a technique by which muscle thin filaments are reconstituted from the recombinant troponin components and the native thin filaments. By this technique, the reconstituted troponin complex is exchanged into the native thin filaments in the presence of 20% glycerol and 0.3 M KCl at pH 6.2. Over 90% of endogenous troponin complex was replaced with the recombinant troponin complex. Structural integrity and Ca(2+)-sensitivity of the reconstituted thin filament prepared by this technique was confirmed by X-ray fiber diffraction measurements and the thin filament activated myosin subfragment 1 ATPase measurements, respectively. PMID: 20064482 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3172317 Fri, 08 Jan 2010 00:00:00 +0100 3172317 http://www.medworm.com/index.php?rid=3172316&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20064483%26dopt%3DAbstract Authors: Atta NF, El-Kady MF, Galal A An interesting electrochemical sensor has been constructed by the electrodeposition of Pd nanoclusters (Pd(nano)) on poly(N-methylpyrrole) (PMPy) film coated Pt electrode. Cyclic voltammetry, electrochemical impedance spectroscopy (EIS) and scanning electron microscope were used to characterize the properties of the modified electrode. It was demonstrated that the electroactivity of the modified electrode depends strongly on the electrosynthesis conditions of the PMPy film and Pd(nano). Moreover, the modified electrode exhibits strong electrocatalytic activity toward the oxidation of a mixture of dopamine (DA), ascorbic acid (AA) and uric acid (UA) with obviously reduction of overpotentials. The simultaneous analysis of this mixture at conventional... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3172316 Fri, 08 Jan 2010 00:00:00 +0100 3172316 http://www.medworm.com/index.php?rid=3164304&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20060801%26dopt%3DAbstract Authors: Ocaña MF, Neubert H An immunoaffinity liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the quantitation of the zinc endopeptidase matrix metalloproteinase 9 (MMP-9) from mouse serum. Sample preparation for the assay included magnetic bead based enrichment using an MMP-9 antibody and was performed in a 96 well plate format using a liquid handling robotic platform. The surrogate peptide GSPLQGPFLTAR derived from MMP-9 by trypsin digestion was monitored using an online capillary flow trap-release chromatography setup incorporating a series trap columns (C18, strong cation exchange and another C18) prior to nanoflow chromatography and nanospray ionization with selected reaction monitoring (SRM) detection. The assay was fit-for-purpose validat... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3164304 Thu, 07 Jan 2010 00:00:00 +0100 3164304 http://www.medworm.com/index.php?rid=3164303&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20060802%26dopt%3DAbstract In this study, intact cells were compared to isolated membranes as receptor support for radioligand receptor-binding assay. Growth hormone secretagogue receptor 1a (GHSR-1a) expressed in HEK293 cells was used as a model of G protein-coupled receptors. Differences between using intact cells in suspension or isolated membranes were evaluated for different aspects of the receptor binding assay: total binding variations while both receptor preparations remain on ice, modifications in incubation conditions, saturation and competition using different agonists. Intact cells are more prone to variability. While under optimized settings both preparation were equivalent, the K(d) value for intact cells was three times higher than using isolated membranes. However, no significant differences were obs... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3164303 Thu, 07 Jan 2010 00:00:00 +0100 3164303 http://www.medworm.com/index.php?rid=3164305&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20060376%26dopt%3DAbstract Authors: Achilonu I, Dean Goldring JP Proteins separated by SDS polyacrylamide gel electrophoresis can be stained with organic dyes, the most popular being Coomassie Brilliant Blue R-250. Coomassie R-250 staining of ovalubumin in an SDS-PAGE gel increased linearly from 2.5 to 60 min. Direct Red 81 and Amido Black staining approached saturation in 10 min. Scatchard analysis showed that the number of Direct Red 81 and Amido Black ligands bound to ovalbumin was four fold higher than that of Coomassie R-250. Direct Red 81 and Amido Black stain proteins in an SDS polyacrylamide electrophoresis gel in 10 minutes. PMID: 20060376 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3164305 Wed, 06 Jan 2010 00:00:00 +0100 3164305 http://www.medworm.com/index.php?rid=3157879&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20056100%26dopt%3DAbstract Authors: Shi C, Gu H, Ma C A novel and sensitive biosensor based on aptamer and pyrene-labeled fluorescent probe for the determination of K(+) was developed. The aptamer was used as molecular recognition element and a partially complementary oligonucleotide with the aptamer was labeled by pyrene moieties at both ends to transduce the binding event of K(+) with aptamer. In the presence of K(+), the complementary oligonucleotides were displaced from aptamers, which was accompanied by excimer fluorescence of pyrenes because the self-hairpin structure of the complementary oligonucleotide brought pyrene moieties into close proximity. However, it gave only monomer emission in the absence of K(+). With optimum conditions, the relative fluorescence intensity of pyrene was proportional to the c... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3157879 Mon, 04 Jan 2010 00:00:00 +0100 3157879 http://www.medworm.com/index.php?rid=3139552&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20045670%26dopt%3DAbstract Authors: Kameshita I, Baba H, Umeda Y, Sueyoshi N We developed a method for detection of phosphatase activity using fluorogenic substrates after polyacrylamide gel electrophoresis. When phosphatases such as Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP), protein phosphatase 2C (PP2C), protein phosphatase 5 (PP5) and alkaline phosphatase were resolved by polyacrylamide gel electrophoresis in the absence of SDS and the gel was incubated with a fluorogenic substrate such as 4-methylumbelliferyl phosphate (MUP), all of these phosphatase activities could be detected in situ. Although 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as well as MUP could be used as a fluorogenic substrate for in-gel assay, MUP exhibited lower background fluorescence. Using this procedure, ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3139552 Thu, 31 Dec 2009 00:00:00 +0100 3139552 http://www.medworm.com/index.php?rid=3139551&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20045671%26dopt%3DAbstract Authors: Roy MC, Ikimura K, Nishino H, Naito T A high recovery microsampling probe based on microdialysis was devised. The new probe showed a high recovery (100%) of peptides in vitro at different perfusion flow rates (0.1-1.0 mul/min). At a high flow rate 1.0 mul/min, a 10-fold increased in recovery of peptides compared to the conventional microdialysis probe was achieved. A probe made of a low molecular weight cutoff membrane is suitable to filter off proteins. The new probe can be a useful tool for high recovery of peptides from living tissues. PMID: 20045671 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3139551 Thu, 31 Dec 2009 00:00:00 +0100 3139551 http://www.medworm.com/index.php?rid=3139553&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20045389%26dopt%3DAbstract Authors: Zhang X, Pan G, Jin X, Tang Q, Wang H, Tu J The present study investigated cDNA chimeras using two closely related members of rice secretory protein gene family as an example. The chimeras detected in initial cDNA products that were amplified using LA Taq polymerase involved two categories: single-site type and multiple-site type with the frequency being about 20% and 3%, respectively. Further investigation revealed that PCR buffer additives and type of DNA polymerase had a major effect on the formation of chimeras in mixed template amplification. Heteroduplex repair by microbial DNA repair systems in cDNA cloning was confirmed to produce the chimeras too, but it was not the major source. PMID: 20045389 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3139553 Wed, 30 Dec 2009 00:00:00 +0100 3139553 http://www.medworm.com/index.php?rid=3137154&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20043864%26dopt%3DAbstract Authors: Lee HJ, Ho MR, Bhuwan M, Hsu CY, Huang MS, Peng HL, Chang HY The manufacturing processes of many electronic and medical products demand the use of high-quality water. Hence the water supply systems for these processes are required to be examined regularly for the presence of microorganisms and microbial biofilms. Among commonly used bacteria detection approaches, the ATP luminescence assay is a rapid, sensitive and easy to perform method. The aim of this study is to investigate whether ATP regeneration from inorganic pyrophosphate, a product of the ATP luminescence assay, can stabilize the bioluminescence signals in ATP detection. ADPglc pyrophosphorylase (AGPPase), which catalyzes the synthesis of ATP from PPi in the presence of ADPglc, was selected because the system yields ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3137154 Mon, 28 Dec 2009 00:00:00 +0100 3137154 http://www.medworm.com/index.php?rid=3129997&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20036206%26dopt%3DAbstract In this study, we found that ceramide extraction mediated by a ceramide trafficking protein (CERT) could be detected as decreasing of the response of surface plasmon resonance (SPR). Based on this finding, we developed a novel real-time assay method that enables quantitative evaluation of the ceramide extraction activity of CERT, using the SPR technique. Performing this SPR-based assay using ceramide-embedded and ceramide-free lipid bilayer as ligands, it allows for the exclusive investigation of ceramide uptake processes, differentiating them from other CERT-membrane binding events. Furthermore, mutagenesis experiments of CERT using this SPR-based assay clearly elucidated whether an amino acid residue plays a role in the ceramide uptake process or the lipid bilayer binding process. This S... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3129997 Wed, 23 Dec 2009 00:00:00 +0100 3129997 http://www.medworm.com/index.php?rid=3129996&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20036207%26dopt%3DAbstract Authors: Zhao C, Hellman LM, Zhan X, Bowman WS, Whiteheart SW, Fried MG The hexahistidine (His(6))/Nickel (II)-Nitrilotriacetic Acid (Ni(2+)-NTA) system is widely used for affinity purification of recombinant proteins. The NTA group has many other applications, including the attachment of chromophores, fluorophores, or nano-gold to His(6)-proteins. Here we explore several applications of the NTA-derivative, (Ni(2+)-NTA)(2)-Cy3. This molecule binds our two model His(6)-proteins, N-ethylmaleimide Sensitive Factor (NSF) and O(6)-alklyguanine-DNA alkyltransferase (AGT), with moderate affinity (K approximately 1.5 x 10(6) M(-1)) and no effect on their activity. Its high specificity makes (Ni(2+)-NTA)(2)-Cy3 ideal for detecting His(6)-proteins in complex mixtures of other proteins, allowing ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3129996 Wed, 23 Dec 2009 00:00:00 +0100 3129996 http://www.medworm.com/index.php?rid=3129995&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20036208%26dopt%3DAbstract Authors: Shlyapnikov YM, Shlyapnikova EA, Morozova TY, Beletsky IP, Morozov VN Efficiency of hybridization analysis with oligonucleotide microarrays depends heavily on the method of detection. Conventional methods based on labeling nucleic acids with fluorescent, chemiluminescent, enzyme or radioactive reporters suffer from a number of serious drawbacks which demand development of new detection techniques. Here we report two new approaches for detection of hybridization with oligonucleotide microarrays employing magnetic beads as active labels. In the first method streptavidin-coated magnetic beads are used to discover biotin-labeled DNA molecules hybridized with arrayed oligonucleotide probes. In the second method biotin-labeled DNA molecules are bound first to the surface of magnetic... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3129995 Wed, 23 Dec 2009 00:00:00 +0100 3129995 http://www.medworm.com/index.php?rid=3129994&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20036209%26dopt%3DAbstract In this study, 13 housekeeping genes, ACTB, B2M, GAPDH, GUSB, HPRT1, HMBS, HSP90AB1, RPL13A, RPS29, RPLP0,PPIA, TBP and TUBA1 were analyzed to determine their applicability for isolated crypt cells, villus cells, de-epithelialized mucosa, and whole mucosa of the mouse small intestine. Using geNorm and NormFinder software, GUSB and TBP were identified as the most stably expressed genes, whereas the expression of the commonly used reference genes GAPDH, B2M, ACTB, and ribosomal protein genes RPL13A, RPS29, RPLP0, were relatively unstable. Thus, this study demonstrates that GUSB and TBP are the optimal reference genes for the normalization of gene expression in the mouse small intestine. PMID: 20036209 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3129994 Wed, 23 Dec 2009 00:00:00 +0100 3129994 http://www.medworm.com/index.php?rid=3129993&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20036210%26dopt%3DAbstract Authors: Rogers JM, Lippert LG, Gai F Fluorescence resonance energy transfer (FRET) provides a powerful means to study protein conformational changes. However, the incorporation of an exogenous FRET pair into a protein could lead to undesirable structural perturbations of the native fold. One of the viable strategies to minimizing such perturbations is to use non-natural amino acid based FRET pairs. Previously we have shown that p-cyanophenylalanine (Phe(CN)) and tryptophan (Trp) constitute such a FRET pair, useful for monitoring protein folding-unfolding transitions. Herein, we further show that 7-azatryptophan (7AW) and 5-hydroxytryptophan (5HW) can also serve as a FRET acceptor to Phe(CN), and the resultant FRET pairs offer certain advantages over Phe(CN)-Trp. For example, the fluor... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3129993 Wed, 23 Dec 2009 00:00:00 +0100 3129993 http://www.medworm.com/index.php?rid=3129998&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20035706%26dopt%3DAbstract Authors: Hung CC, Chen SU, Lin SY, Fang MY, Chang LJ, Tsai YY, Lin LT, Yang YS, Lee CN, Su YN Preimplantation genetic diagnosis (PGD) is employed increasingly to allow transfer of embryos to the uterus in assisted reproduction procedures. There are three stages of biopsy: polar bodies, one or two blastomere from the cleavage-stage embryos and trophectoderm cells (around 5 cells) from the blastocyst-stage embryos. Validation of PCR-based assays are challenging because only limited genetic material can be obtained for PGD. In the present study, we modified a valid single-cell PCR protocol for PGD using real-time PCR assay with fluorescence resonance energy transfer (FRET) hybridization probes followed by melting curve analysis. We optimized and clinically applied the protocol permitting ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3129998 Tue, 22 Dec 2009 00:00:00 +0100 3129998 http://www.medworm.com/index.php?rid=3122555&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20034461%26dopt%3DAbstract Authors: Strucksberg KH, Tangavelou K, Schröder R, Clemen CS The ubiquitin-proteasome-system (UPS) is a major degradation system for regulatory and mis-folded proteins. UPS-function has been implicated to exert a central role in the pathogenesis of various human diseases. Since biochemical analyses are often hampered by the amount of available diseased tissue, we report on the establishment and validation of a luminescence-based proteasomal activity assay applicable to five-milligram quantities of skeletal muscle. We demonstrate that the specific proteasomal activity differs in individual muscle groups and decreases with ageing. These findings warrant the use of appropriate controls and a careful interpretation of results in mammalian skeletal muscle pathologies. PMID: 2003446... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3122555 Mon, 21 Dec 2009 00:00:00 +0100 3122555 http://www.medworm.com/index.php?rid=3112122&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20026293%26dopt%3DAbstract Authors: Zhang H, Il'yasova D, Sztaray J, Young SP, Wang F, Millington DS F2-isoprostanes are useful biomarkers of oxidative status in humans. We developed an ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify 2,3-dinor-8-iso prostaglandin F(2alpha), an urinary metabolite of 8-iso-prostaglandin F(2alpha.) Urine was purified by solid phase extraction and analyzed by UPLC-MS/MS with negative ion-electrospray ionization. The method was robust with a mean inaccuracy of 9%, interday and intraday imprecision of 7.5% or lower, and a lower limit of quantification of 0.5 mug/L, equivalent to 0.04 pmol injected onto the column. An analysis time of 6 minutes was shorter than previously published methods and amenable to large studies. PMID: 200262... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3112122 Fri, 18 Dec 2009 00:00:00 +0100 3112122 http://www.medworm.com/index.php?rid=3112121&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20026294%26dopt%3DAbstract Authors: Marczylo TH, Lam PM, Amoako AA, Konje JC Anandamide (N-arachidonoylethanolamide) a bioactive lipid is reported to play a role in pregnancy maintenance and parturition. Our aims were 1. Evaluate AEA levels at the human maternal: fetal interface and 2. Validate the use of solid-phase extraction of AEA from tissues. AEA was analyzed in cord and maternal blood, amniotic fluid, placenta and fetal membranes collected during Caesarean section (n=14. Extraction efficiencies were 42% and 36% for the placenta and the fetal membranes, respectively. Tissue AEA was quantified using an isotope-dilution method and UPLC-ESI-MS/MS giving intra- and inter-day variability for tissues spiked with 0.2, 1 and 5 pmol/g AEA of less than 12%. Accuracy for these spiked samples was between 95-103% for f... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3112121 Fri, 18 Dec 2009 00:00:00 +0100 3112121 http://www.medworm.com/index.php?rid=3112120&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20026295%26dopt%3DAbstract Authors: Lopez MM, Atherton AA, Tong WG Nonlinear laser wave mixing is presented as an ultrasensitive absorption-based method for the detection of proteins and antibodies using a non-fluorescing chromophore label, Coomassie Brilliant Blue (CBB). The complexes are flowed through a 150 mum i.d. capillary cell and detected using a low-power He-Ne laser. The wave-mixing signal is detected after 10 minutes of room-temperature incubation for the antibody complex and 18 minutes for the protein complex. All solutions are prepared in an aqueous buffer without the addition of organic modifiers. Concentration detection limits of 3.4 x 10(-19) M and 6.4 x 10(-14) M (S/N 2) are determined for bovine serum albumin (BSA) and human Papillomavirus (HPV) antibody, respectively. Based on the small laser ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3112120 Fri, 18 Dec 2009 00:00:00 +0100 3112120 http://www.medworm.com/index.php?rid=3112119&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20026296%26dopt%3DAbstract In conclusion, the BW extraction is recommended for analysis of skeletal muscle metabolome, except for creatine-P which was poorly recovered with this technique. PMID: 20026296 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3112119 Fri, 18 Dec 2009 00:00:00 +0100 3112119 http://www.medworm.com/index.php?rid=3112118&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20026297%26dopt%3DAbstract This report describes the application of tree diagrams and bootstrapping techniques for an improved quantitative analysis of metabolic PCA data clustering. Our PCAtoTree program creates a distance matrix with 100 bootstrap steps that describes the separation of all clusters in a metabolic dataset. Using accepted phylogenetic software, the distance matrix resulting from the various metabolic states is organized into a phylogenetic-like tree format, where bootstrap values 50 indicate a statistically relevant branch separation. PCAtoTree analysis of two previously published data sets demonstrates the improved resolution of metabolic state differences using tree diagrams. In addition, for metabolomic studies of large numbers of different metabolic states, the tree format provides a better desc... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3112118 Fri, 18 Dec 2009 00:00:00 +0100 3112118 http://www.medworm.com/index.php?rid=3112123&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20026005%26dopt%3DAbstract Authors: Coleman DJ, Kuntz DA, Venkatesan M, Cook GM, Williamson SP, Rose DR, Naleway JJ A simple and reliable continuous assay for measurement of alpha-mannosidase activity is described and demonstrated for analysis with two recombinant human enzymes using the new substrate resorufin alpha-D-mannopyranoside (Res-Man). The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585 nm with excitation at 571 nm and has a pKa of 5.8, allowing continuous measurement of fluorescence turnover at or near physiological pH values for human lysosomal and Drosophila Golgi alpha-mannosidases. The assay performed using recombinant Drosophila Golgi alpha-mannosidase (dGMII) has been shown to give the kinetic parameters K(m) of 200 muM and V(max) of 11 nmol/min per nmol dGMII. Metho... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3112123 Wed, 16 Dec 2009 00:00:00 +0100 3112123 http://www.medworm.com/index.php?rid=3112124&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20025845%26dopt%3DAbstract Authors: Zorrilla S, Garzón B, Pérez-Sala D Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily involved in insulin sensitization, atherosclerosis, inflammation and carcinogenesis. PPARgamma transcriptional activity is modulated by specific ligands which promote conformational changes allowing interaction with coactivators. Here we show that the fluorophore 1-anilinonaphthalene-8-sulfonic acid (ANS) binds to PPARgamma-LBD, displaying negligible interaction with other nuclear receptors like PPARalpha or RXRalpha. ANS binding is competed by PPARgamma agonists, such as rosiglitazone, 15-deoxy-Delta(12,14)-Prostaglandin J(2) (15d-PGJ(2)) and CAY10410. Moreover, the affinity of PPARgamma for these ligands, determined through... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3112124 Tue, 15 Dec 2009 00:00:00 +0100 3112124 http://www.medworm.com/index.php?rid=3105487&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20018158%26dopt%3DAbstract We report here the solubilization of the human NPFF(2) receptor expressed in Chinese Hamster Ovary (CHO) cells by the zwitterionic detergent CHAPS. CHAPS solubilization resulted in the abolishment of specific agonist binding activity, which was restored by a PEG precipitation method. Reincorporation after the precipitation step into liposomes made of endogenous lipids issued from CHO membranes or exogenous lipids significantly enhanced the specific agonist binding activity and G-protein coupling. This method of solubilization and lipid reconstitution could be useful for studies of NPFF receptors. PMID: 20018158 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3105487 Mon, 14 Dec 2009 00:00:00 +0100 3105487 http://www.medworm.com/index.php?rid=3105486&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20018159%26dopt%3DAbstract This study sought to determine whether the quality of enzyme preparations can be determined from their melting curves, which may easily be obtained using a fluorescent probe and a standard RT-PCR machine. Thermal melt data on 31 recombinant enzymes from Plasmodium parasites were acquired by incrementally heating them to 90 degrees C and measuring unfolding with a fluorescent dye; activity assays specific to each enzyme were also performed. Four of the enzymes were denatured to varying degrees with heat and SDS prior to the thermal melt and activity assays. In general, melting curve quality correlated with enzyme activity; enzymes with high-quality curves were found almost uniformly to be active, while those with lower-quality curves were more varied in their catalytic performance. Inspecti... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3105486 Mon, 14 Dec 2009 00:00:00 +0100 3105486 http://www.medworm.com/index.php?rid=3105485&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20018160%26dopt%3DAbstract Authors: Kotarek JA, Moss MA Evidence that membrane-associated amyloid aggregate growth can impart membrane damage represents one possible mechanism for the neurodegeneration associated with deposited amyloid- beta protein (A beta) aggregates in the brains of Alzheimer's disease (AD) patients. This potential pathogenic event necessitates an understanding of the impact that cellular membrane composition may have upon A beta aggregate growth.In the current study, a quartz crystal microbalance (QCM) was employed to examine the growth of A beta(1-40) aggregation intermediates upon supported phospholipid bilayers (SPBs) assembled at the crystal surface. These surface-specific measurements illustrate that zwitterionic SPBs selectively bind aggregated but not monomeric protein, and these boun... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3105485 Mon, 14 Dec 2009 00:00:00 +0100 3105485 http://www.medworm.com/index.php?rid=3105484&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20018161%26dopt%3DAbstract Authors: Matsumoto H, Akita K, Sakai R, Shimomura Y We have developed a highly sensitive, non-radioisotope method for the measurement of branched-chain alpha-keto acid dehydrogenase complex (BCKDC) activity using alpha-keto[1-(13)C]isocaproate ([1-(13)C]KIC) as substrate. The enzyme reaction was performed in a 10-mL test tube. After incubation of the reaction mixture (1 mL), the reaction was terminated by acidification, followed by addition of internal standard (3.5 mumol K(2)CO(3)) for calculation of (13)CO(2). The (13)CO(2) released to the gas phase in the test tube was calculated from the molar ratio of (13)CO(2)/(12)CO(2) assayed by gas chromatography isotope ratio mass spectrometry. A distinct linear relationship between (13)CO(2) production rate and BCKDC activity was obtained wh... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3105484 Mon, 14 Dec 2009 00:00:00 +0100 3105484 http://www.medworm.com/index.php?rid=3105483&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20018162%26dopt%3DAbstract Authors: Li J, Wang Z, He G, Zhang W, Qian A, Shang P Normalization is essential to the in-cell western (ICW) assay, a near-infrared immunocyto-blot for protein analysis. Here we report that cells reacted with glutaraldehyde fluoresced in the near-infrared region of the spectrum, and the intensity of fluorescence was directly proportional to cell number over a range of 3,125 to 100,000 per well. We took advantage of this property to develop a method for quantification of cells, and applied it to ICW assay for normalization. The application of glutaraldehyde may make the ICW assay more popular due to the reduced cost and simplified procedure. PMID: 20018162 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3105483 Mon, 14 Dec 2009 00:00:00 +0100 3105483 http://www.medworm.com/index.php?rid=3105482&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20018163%26dopt%3DAbstract Authors: Ruan BH, Cole DC, Wu P, Quazi A, Page K, Wright JF, Huang N, Stock JR, Nocka K, Aulabaugh A, Krykbaev R, Fitz LJ, Wolfman NM, Fleming ML Vanin-1 is a pantetheinase that catalyzes the hydrolysis of pantetheine to produce pantothenic acid (Vitamin B5) and cysteamine. Reported here is a highly sensitive fluorescent assay using a novel fluorescently-labeled pantothenate derivative. The assay has been utilized for characterization of a soluble version of human vanin-1 recombinant protein, identification and characterization of hits from HTS screening, and quantification of vanin pantothenase activity in cell lines and tissues. Under optimized assay conditions, we quantified vanin pantothenase activity in tissue lysate and found low activity in lung and liver but high activity in ki... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3105482 Mon, 14 Dec 2009 00:00:00 +0100 3105482 http://www.medworm.com/index.php?rid=3105481&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20018164%26dopt%3DAbstract Authors: Johnson RM, Allen C, Melman SD, Waller A, Young SM, Sklar LA, Parra KJ Fluorescence intensity of the pH-sensitive carboxyfluorescein derivative BCECF was monitored by high throughput flow cytometry in living yeast cells. We measured fluorescence intensity of BCECF trapped in yeast vacuoles, acidic compartments equivalent to lysosomes where V-ATPases are abundant. Because V-ATPases maintain a low pH in the vacuolar lumen, V-ATPase inhibition by concanamycin A alkalinized the vacuole and increased BCECF fluorescence. Likewise, V-ATPase deficient mutant cells had greater fluorescence intensity than wild-type cells. Thus, we detected an increase of fluorescence intensity after short-term and long-term inhibition of V-ATPase function. We used yeast cells loaded with BCECF to screen... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3105481 Mon, 14 Dec 2009 00:00:00 +0100 3105481 http://www.medworm.com/index.php?rid=3105479&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20018165%26dopt%3DAbstract In conclusion, this study emphasises the significance of ABC-transporters for the efficacy of selection processes. Consciousness of the results is supposed to guide the molecular biologist to the right choice of adequate experimental conditions for successful selection of genetically engineered eucaryotic cells. PMID: 20018165 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3105479 Mon, 14 Dec 2009 00:00:00 +0100 3105479 http://www.medworm.com/index.php?rid=3105478&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20018166%26dopt%3DAbstract Authors: Wang Z, Zhang Z, McCallum SA, Linhardt RJ Quantification of heparosan produced from the E. coli K5 strain typically relies on chromatography following extensive purification. Such purification steps are both time-consuming and can result in sample loss and thus may not accurately reflect the amount of heparosan in the original mixture. In the current study, a simple, sensitive 1H-NMR quantification method is described that directly quantifies heparosan K5 polysaccharide present in E. coli fermentation supernatant. PMID: 20018166 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3105478 Mon, 14 Dec 2009 00:00:00 +0100 3105478 http://www.medworm.com/index.php?rid=3105488&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20015447%26dopt%3DAbstract Authors: Holtfrerich A, Makharadze T, Lehr M A fluorescent assay for the evaluation of inhibitors of monoacylglycerol lipase (MAGL) is described. 1,3-Dihydroxypropan-2-yl 4-pyren-1-ylbutanoate was designed and synthesized as novel fluorogenic substrate. Activity of human recombinant MAGL was determined in presence of the surfactant Triton X-100 without further sample clean-up by measuring the amount of 4-pyren-1-ylbutanoic acid released by the enzyme with reversed-phase HPLC and fluorescence detection. The known covalent binding MAGL inhibitors methyl arachidonyl fluorophosphonate (MAFP), JZL184 and CAY10499 were used to validate the test system. Applying an incubation time of 15 min, the IC(50) values obtained for these compounds were 0.16 muM, 3.7 muM and 1.1 muM, respectively. A pro... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3105488 Sun, 13 Dec 2009 00:00:00 +0100 3105488 http://www.medworm.com/index.php?rid=3072405&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19995543%26dopt%3DAbstract Authors: He F, Becker GW, Litowski JR, Narhi LO, Brems DN, Razinkov VI We propose a new method to measure the viscosity of concentrated protein solutions in a high throughput format. This method measures the apparent hydrodynamic radius of polystyrene beads with known sizes using a dynamic light scattering (DLS) system with a microplate reader. Glycerol solution viscosities obtained by the DLS method were in good agreement with those reported in the literature. Viscosity of the solutions of two monoclonal antibody molecules was acquired using both DLS and cone-and-plate techniques, and the results were comparable. The DLS method described here has the potential to be used in many aspects of protein characterization. PMID: 19995543 [PubMed - as supplied by publisher] (Source: Analyt... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3072405 Sat, 05 Dec 2009 00:00:00 +0100 3072405 http://www.medworm.com/index.php?rid=3072404&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19995544%26dopt%3DAbstract Authors: Lihme A, Hansen MB, Andersen IV, Burnouf T Current plasma fractionation technology combines ethanol precipitation with packed bed chromatography. We have developed a novel core fractionation process comprising 5 expanded bed adsorption (EBA) chromatographic steps on high-density modified agarose/tungsten carbide beads. Plasma was first chromatographed on two DEAE-tungsten carbide agarose adsorbents (respective mean particle diameters of d(v)(0.5) = 190 mum and d(v)(0.5) = 37 mum) to isolate at 50-80% recovery a fraction containing 4-7 IU/ml of Factor II (FII), FIX, and FX (specific activity over 1 IU/mg) and another, enriched in FVIII and von Willebrand factor (VWF) (about 1 IU/ml and 0.6 IU/mg), respectively. The flow-through was adsorbed on 4% agarose-10% tungsten carbide be... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3072404 Sat, 05 Dec 2009 00:00:00 +0100 3072404 http://www.medworm.com/index.php?rid=3072403&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19995545%26dopt%3DAbstract Authors: Hu CM, Chang ZF Blocking human thymidylate kinase (TMPK) function has a chemosensitization effect in anticancer treatment. However, a rapid and sensitive TMPK activity assay method suitable for inhibitor screening has been lacking. Here, we designed a luciferase-coupled TMPK assay, in which luminescence emission is proportional to the magnitude of TMPK inhibition. The advantages of using this new method over the conventional NADH-coupling method in screening inhibitor include low cost, low limit in detecting inhibitory signal, more accurate, and devoid of interference due to compound absorbance at 340 nm. PMID: 19995545 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3072403 Sat, 05 Dec 2009 00:00:00 +0100 3072403 http://www.medworm.com/index.php?rid=3066738&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19962366%26dopt%3DAbstract Authors: Anderson H, Myrskog A, Dunér G, Ingemarsson B, Aastrup T, Pei Z The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We have shown that successful immobilization is highly dependent on surface pK(a), antibody pI and pH of immobilization buffer. By use of EDC/sulfo-NHS activation reagents, the effect of the intrinsic surface pK(a) is avoided and immobilization also at very low pH has been made possible which is of importance for immobilization of acidic proteins. Antigen binding capacity as a function of immobilization pH was studie... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3066738 Wed, 02 Dec 2009 00:00:00 +0100 3066738 http://www.medworm.com/index.php?rid=3066737&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19962367%26dopt%3DAbstract Authors: Hall D Observations of the motion of individual molecules in the membrane of a number of different cell types has led to the suggestion that the outer membrane of many eukaryotic cells may be effectively partitioned into micro-domains. A major cause of this suggested partitioning is believed due to the direct/indirect association of the cytosolic face of the cell membrane with the cortical cytoskeleton. Such intimate association is thought to introduce effective hydrodynamic barriers into the membrane that are capable of frustrating molecular Brownian motion over distance scales greater than the average size of the compartment. To date the standard analytical method for deducing compartment characteristics has relied upon observing the random walk behaviour of a labelled lipid... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3066737 Wed, 02 Dec 2009 00:00:00 +0100 3066737 http://www.medworm.com/index.php?rid=3066750&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19961824%26dopt%3DAbstract Authors: Lee HH, Lee YJ, Chao MC The 3.2-kb TaqI-produced fragment of the CYP21A1P pseudogene and the 3.7-kb TaqI-produced fragment of the functional CYP21A2 gene exist on chromosome 6p21.3. We used the PCR product and Southern blot method with TaqI endonuclease digestion to identify a chimeric-RCCX module in 2 unrelated CAH patients with congenital adrenal hyperplasia (CAH). After TaqI cleavage, the PCR product analysis revealed that patient 1 with the chimeric CYP21A1P/CYP21A2 gene in 1 allele and IVS2-12A/C>G in combination with the 707-714del mutation in the other allele produced a configuration of 3.2- and 2.4-kb fragments. Patient 2 who carried IVS2-12A/C>G in combination with the 707-714del mutation in 1 allele and the chimeric TNXA/TNXB gene in the other allele presented ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3066750 Tue, 01 Dec 2009 00:00:00 +0100 3066750 http://www.medworm.com/index.php?rid=3066748&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19961825%26dopt%3DAbstract Authors: Chen G, Kronenberger P, Umelo IA, Teugels E, Grève JD A simple and sensitive real time Reverse Transcriptase-quantitative PCR (RT-qPCR) was developed to quantify T790M mutant transcripts in a wild type (wt) EGFR background. The assay is based on 3 unmodified oligonucleotides and both SYBR green and a Taqman probe can be used. To increase the discrimination between mutant and wt signals, ARMS and LNA primers were tested, but a benefit was only observed with plasmids, not with cellular cDNA. The RT-qPCR assay using transcript-specific primers can detect as few as 1% T790M transcripts in a wt background, and will therefore be useful in RNA interference studies specifically targeting mutant RNA. PMID: 19961825 [PubMed - as supplied by publisher] (Source: Analytical Bioche... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3066748 Tue, 01 Dec 2009 00:00:00 +0100 3066748 http://www.medworm.com/index.php?rid=3066746&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19961826%26dopt%3DAbstract Authors: Huang Y, Zhao S, Shi M, Liu YM A homogenous chemiluminescent immunoassay of thyroxine (T4) enhanced by microchip electrophoresis separation has been developed. The method deployed the competitive immunoreaction of T4 and horseradish peroxidase (HRP)-labeled T4 (HRP-T4) with anti-T4 mouse monoclonal antibody (Ab). HRP-T4 and the HRP-T4-Ab complex were separated and quantified by using microchip electrophoresis (MCE) with chemiluminescence (CL) detection. Highly sensitive CL detection was achieved by means of HPR-catalyzed luminol-H(2)O(2) reaction. Due to the effective MCE separation, the CL analytical signal was less prone to sample matrix interference. Under the selected assay conditions, the MCE separation was accomplished within 60 sec. The linear range for T4 were 5 -250 n... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3066746 Tue, 01 Dec 2009 00:00:00 +0100 3066746 http://www.medworm.com/index.php?rid=3066739&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19961827%26dopt%3DAbstract Authors: Khan F, Saxl TE, Pickup JC We synthesized mutants of glucose/galactose binding protein (GBP) labelled with the environmentally sensitive fluorophore Badan, with the aim of producing a fluorescence-based glucose sensing system with an operating range compatible with continuous glucose monitoring in patients with diabetes mellitus. From five mutants tested, the triple mutant H152C/A213/L238S-Badan showed a large (200%) maximal increase in fluorescence intensity on glucose addition, with a binding constant (K(d)) of 11 mM, an operating range of approximately 1-100 mM and similar responses in buffer and serum. The mean fluorescence lifetime of this mutant also increased by 70% on glucose addition. We conclude that GBP mutant H152C/A213/L238S, when labelled with Badan, is suitable ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3066739 Tue, 01 Dec 2009 00:00:00 +0100 3066739 http://www.medworm.com/index.php?rid=3057203&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19951694%26dopt%3DAbstract Authors: Yan J, Wang H, Jain N, Toxavidis V, Tigges J, Yang H, Yue G, Gao W 2A peptide sequences are now being widely used to construct multi-cistronic expression vectors. It is suggested that when only the first 2A-linked protein bears a signal sequence, the signal-less protein(s) downstream of 2A can also be translocated into the mammalian ER system through a "slipstreaming" mechanism. By using flow cytometry and cell surface CD90 as a localization indicator, we show here that "slipstreaming" translocation does not occur in mammalian cells, i.e., the second protein downstream of 2A still requires signal sequence for secretary or membrane-anchored expression. PMID: 19951694 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3057203 Sat, 28 Nov 2009 00:00:00 +0100 3057203 http://www.medworm.com/index.php?rid=3049023&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19944666%26dopt%3DAbstract Authors: Shin D, Nam M, Yoon Y, Kim M Peptide nucleic acid (PNA) is a chimeric oligonucleotide with nucleotide-derived bases and a peptide backbone. Compared with natural nucleotides, PNA has several advantages, including improved stability and high sequence discrimination during duplex formation. Despite its potential for therapeutic application, analysis technologies have not been generalized mainly due to ambiguous physiochemical properties resembling those of nucleic acids as well as protein. Here, we present a PNA detection method: SDS-PAGE followed by electro-transfer to a Western-blotting membrane and then hybridization with a radio-labeled oligonucleotide probe. This method is useful for evaluating the quality of synthetic PNA and determining its intracellular localization. ... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3049023 Wed, 25 Nov 2009 00:00:00 +0100 3049023 http://www.medworm.com/index.php?rid=3049025&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19944058%26dopt%3DAbstract Authors: Heredia NJ, Beer NR, Hara CA, Hiddessen AL, Bailey CG A double transposition technique is presented that inserts two different transposons into target DNA to act as priming sites for amplifying the region between the two transposons for sequencing applications. Unlike some current sequencing approaches, the genome of the unknown target remains intact in this method. The transposition reaction, DNA repair, and subsequent sequencing were performed entirely in vitro, without the need for transformation into bacteria, and resulted in sequence homology with the plasmid DNA target. This approach can reduce the time required for the assay by over a day compared to standard techniques and reduces the number of required enzymatic steps. In addition, the in vitro method enables transpos... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3049025 Tue, 24 Nov 2009 00:00:00 +0100 3049025 http://www.medworm.com/index.php?rid=3049024&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19944059%26dopt%3DAbstract We report herein a simple, biomolecular friendly protocol for the fabrication of a hydroxyapatite nanowires array (HANWA) biosensor of spatial positioning, large surface area and abundant adsorbing sites and its application to cyanide sensing. The fabrication of HANWA is performed by template-assisted electrodeposition. The well-aligned hydroxyapatite nanoarray is composed of vertical nanowires with the diameter of about 200 nm and the average length of 1 mum. The electrochemical biosensor for the determination of cyanide through its inhibitory effect on horseradish peroxidase (HRP) encapsulated by chitosan (CHIT) on the platform of hydroxyapatite nanowires array is demonstrated. The present organic-inorganic hybrid nanostructure provides excellent enzyme-substrate contact with enzyme acti... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3049024 Tue, 24 Nov 2009 00:00:00 +0100 3049024 http://www.medworm.com/index.php?rid=3036438&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19941831%26dopt%3DAbstract We describe a general protocol for preparing protein-containing biofluids for (1)H-NMR metabolomics studies. In this protocol, untreated samples are diluted in deuterated solvents to precipitate proteins and recover metabolites quantitated relative to standard reference compounds such as 3-trimethylsilylpropionic acid (TSP) and 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS). The efficacy of this protocol was tested using a bovine serum albumin/metabolite mix and human serum samples. This sample-preparation method can be readily applied to any protein-containing biofluid for (1)H-NMR studies. PMID: 19941831 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3036438 Mon, 23 Nov 2009 00:00:00 +0100 3036438 http://www.medworm.com/index.php?rid=3031444&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19932676%26dopt%3DAbstract Authors: Wang AJ, Vainikka K, Witos J, D'Ulivo L, Cilpa G, Kovanen PT, Oörni K, Jauhiainen M, Riekkola ML Human plasma lipoproteins have strong hydrophobic interactions with steroids and their fatty acyl derivatives such as estradiol fatty acyl esters. In this work, affinity capillary electrophoresis with the partial-filling technique was applied to study the hydrophobic interactions between lipoproteins, which are nanometer-sized particles, and non-conjugated steroids. The capillaries were first rinsed with one of two novel poly(vinylpyrrolidone)(PVP)-based cationic copolymers that were strongly adsorbed onto the fused-silica surface via electrostatic interactions. This surface treatment greatly suppressed the adsorption of lipoproteins. Low-density (LDL) and high-density lipopro... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3031444 Fri, 20 Nov 2009 00:00:00 +0100 3031444 http://www.medworm.com/index.php?rid=3031443&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19932677%26dopt%3DAbstract Authors: Kumar SS, Chouhan RS, Thakur MS Vitamin B(12) is an organic compound containing cobalt and essential nutrient for all cell development and human growth. The daily requirements of vitamin B(12) are very low and deficiencies reported to be at picogram level, thus it necessitates detecting vitamin B(12) at high sensitivity in biological samples. It is also reported that several functional groups in the vitamin B(12) and analogs make more difficult to analyze in biological samples for routine analysis, as analogs are not useful for human metabolism. Many methods have been reported for its analysis like radioisotope, high performance liquid chromatography (HPLC), spectrophotometry, fluorimetric assay, capillary electrophoresis (CE) and atomic absorption spectrometry (AAS). These co... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3031443 Fri, 20 Nov 2009 00:00:00 +0100 3031443 http://www.medworm.com/index.php?rid=3031442&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19932678%26dopt%3DAbstract Authors: Kim SE, Gorrell A, Rader SD, Lee CH Apurinic/apyrimidinic endonuclease 1 (APE1) is a multi-functional enzyme with a well-established abasic DNA cleaving activity in the base excision DNA repair pathway, and in providing redox activity to several well known transcription factors. APE1 has recently been shown to cleave at UA, CA, and UG sites of c-myc RNA in vitro and regulates c-myc mRNA in cells. To further understand this new endoribonuclease activity of APE1, we have developed an accurate, sensitive and rapid real-time endonuclease assay based on a fluorogenic oligodeoxynucleotide substrate with a single ribonucleotide. Using this substrate, we carried out the first kinetic analysis of APE1 endoribonuclease activity. We found that the purified native APE1 cleaves the fluorog... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3031442 Fri, 20 Nov 2009 00:00:00 +0100 3031442 http://www.medworm.com/index.php?rid=3031450&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19932072%26dopt%3DAbstract Authors: Brown JW, McKnight CJ F-actin binding constants are traditionally determined by centrifugal cosedimentation with actin microfilaments, where bound protein is separated from actin with SDS-PAGE and quantitated using densitometry. Here, we demonstrate that UV quantitation of reverse-phase HPLC separated proteins provides increased accuracy and sensitivity, can be fully automated, and allows one to perform F-actin competition assays on similar sized proteins. PMID: 19932072 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry) Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3031450 Thu, 19 Nov 2009 00:00:00 +0100 3031450 http://www.medworm.com/index.php?rid=3031449&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19932073%26dopt%3DAbstract Authors: Viltrop T, Krjutškov K, Palta P, Metspalu A We compared six DNA extraction methods for obtaining DNA from whole blood and saliva for use in multiplex PCR assays. The aim was to evaluate saliva sampling as an alternative to blood sampling to obtain DNA for molecular diagnostics, genetic genealogy and research purposes. The DNA quantity, DNA purity (A(260/280)), PCR inhibition ratio and mitochondrial DNA/genomic DNA ratio were measured in order to compare the extraction methods. The different extraction methods resulted in variable DNA quantity and purity but there were no significant differences in the efficiency of multiplex PCR and oligomicroarray signals after single base extension on APEX-2. PMID: 19932073 [PubMed - as supplied by publisher] (Source: Analytical Bi... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3031449 Thu, 19 Nov 2009 00:00:00 +0100 3031449 http://www.medworm.com/index.php?rid=3031448&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19932074%26dopt%3DAbstract Authors: Schuhmacher T, Lemuth K, Hardiman T, Vacun G, Reuss M, Siemann-Herzberg M Current mRNA quantification methods are sophisticated tools for the analysis of gene regulation. However, these methods are not suitable for more complex quantitative approaches such as the mathematical modeling of the in vivo regulation of transcription where dynamic cytosolic mRNA concentrations need to be taken into consideration. In the present paper, the "standard curve method" for quantitative reverse transcription real-time PCR (qRT-PCR) was extended by including an internal RNA standard. This standard enables transcript losses that occur during the process as well as variations resulting from non-quantitative processes to be accounted for. The use of an internal standard yielded transcript concen... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3031448 Thu, 19 Nov 2009 00:00:00 +0100 3031448 http://www.medworm.com/index.php?rid=3031447&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19932075%26dopt%3DAbstract Authors: Campbell FM, Rucklidge GJ, Reid MD, Cantlay L, Robins SP Protein-bound pyrroles are a sign of oxidative damage. Here we report a specific method for detecting pyrrole-containing proteins using biotin-labeled Ehrlich's reagent (ER-B). After treatment of either human serum or isolated human serum proteins with various oxidising agents, damaged, biotin-labeled components could be detected by blotting. Combining the use of ER-B with proteomic techniques allowed human serum proteins susceptible to oxidative damage to be detected and then identified by LC/MS/MS. Identification of such proteins in different human conditions such as obesity, diabetes and cardiovascular disease should lead to the discovery of new biomarkers and the development of specific assays to monitor health statu... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3031447 Thu, 19 Nov 2009 00:00:00 +0100 3031447 http://www.medworm.com/index.php?rid=3031446&cid=s_34389_60_f&fid=34389&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19932076%26dopt%3DAbstract Authors: Grzyska PK, Hausinger RP, Proshlyakov DA The addition of divalent metal ions or substrate taurine to TauD, an alpha-ketoglutarate dependent dioxygenase, alters its UV absorption, as clearly observed by monitoring the protein's difference spectra. Binding of metal ions leads to a decrease in absorption at approximately 297 nm and modulation of other features. A separate signature with enhanced absorption at approximately 295 nm is identified for binding of taurine. These narrow ( approximately 700 cm(-1)) and intense ( approximately 0.5 mM(-1) cm(-1)) spectral changes are attributed to ligand-induced protein conformational changes affecting the environment of aromatic residues. The changes in the UV difference spectra were exploited to assess directly the thermodynamics and kin... Analytical Biochemistry journals http://www.medworm.com/rss/comments.php?id=3031446 Thu, 19 Nov 2009 00:00:00 +0100 3031446