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876 records returned

A method for enzyme quenching in microbial metabolome analysis successfully applied to gram-positive and gram-negative bacteria and yeast.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Although microbial metabolome analysis has now become a widely used method, no generally applicable quenching method has been published so far. Either the methods were established for only one defined organism or the metabolite coverage was quite low. In the current work, a novel, reliable, and robust quenching method for different types of organisms is described. Compared with the commonly used quenching procedure with 60% methanol (-50 degrees C), we obtained improved results for three examined organisms with different cell wall and membrane structures using a 40% ethanol/0.8% sodium chloride solution (-20 degrees C)...
Source: Analytical Biochemistry - November 15, 2009 Category: Biochemistry Authors: Spura J, Reimer LC, Wieloch P, Schreiber K, Buchinger S, Schomburg D Tags: Anal Biochem Source Type: journals

Flow injection analysis electrospray ionization mass spectrometry for high-throughput screening of a gene library for amidase activity.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In high-throughput screening of gene and mutant libraries, high analysis speeds and short method development times are important factors. Mass spectrometry (MS) is considered to be a generic analytical technique with a relatively short development time. Furthermore, when applying flow injection analysis (FIA) for sample introduction, the requirements for high throughput are met. In this work, the use of a single quadrupole electrospray MS instrument for assaying amidase activity in a gene library is demonstrated. The desired selectivity for measuring the amino acid, the reaction product of the amidase reaction, in the ...
Source: Analytical Biochemistry - November 15, 2009 Category: Biochemistry Authors: Henderickx HJ, Raemakers-Franken PC, van der Wal S, de Koster CG, Duchateau AL, Sonke T Tags: Anal Biochem Source Type: journals

Detecting low-abundance vasoactive peptides in plasma: progress toward absolute quantitation using nano liquid chromatography-mass spectrometry.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present novel data using nano liquid chromatography-mass spectrometry (LC-MS) to simultaneously measure a select group of vasoactive peptides (angiotensin, bradykinin, and related hormones) in 50-microl plasma samples, enabling repeated sampling in rodent models. By chromatographically resolving target peptides and using multiple reaction monitoring to enhance MS sensitivity, linear responses down to 10(-17) mol were achieved. Purification of plasma peptides by either methanol precipitation or off-line high-performance liquid chromatography (HPLC) fractionation enabled the detection of endogenous peptides and revealed a...
Source: Analytical Biochemistry - November 15, 2009 Category: Biochemistry Authors: Lortie M, Bark S, Blantz R, Hook V Tags: Anal Biochem Source Type: journals

Progressive degradation of serum samples limits proteomic biomarker discovery.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Preanalytical variables play a key role in discovery of biomarkers. Although the effect of several preanalytical variables on the mass spectral profiles has been studied extensively, little is known about long-term storage of serum samples. This is important because samples used in case-control or epidemiological studies are usually stored for a long time before analysis. Here we evaluated long-term storage effects on mass spectral peak patterns of serum peptides extracted using weak cation exchange magnetic beads. For this, 20 serum samples stored at -80 degrees C were divided equally into two groups based on their st...
Source: Analytical Biochemistry - November 15, 2009 Category: Biochemistry Authors: Ahmad S, Sundaramoorthy E, Arora R, Sen S, Karthikeyan G, Sengupta S Tags: Anal Biochem Source Type: journals

Fluorescence assay of non-transferrin-bound iron in thalassemic sera using bacterial siderophore.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Transfusional iron overload associated with thalassemia leads to the appearance of non-transferrin-bound iron (NTBI) in blood that is toxic and causes morbidity and mortality via tissue damage. Hence, a highly sensitive and accurate assay of NTBI, with broad clinical application in both diagnosis and validation of treatment regimens for iron overload, is important. An assay based on iron chelation by a high-affinity siderophore, azotobactin, has been developed. The steps consist of blocking of native apotransferrin iron binding sites, mobilization of NTBI, ultrafiltration of all serum proteins, and finally the addition...
Source: Analytical Biochemistry - November 15, 2009 Category: Biochemistry Authors: Sharma M, Saxena R, Gohil NK Tags: Anal Biochem Source Type: journals

A convenient plasmid-based system containing three reporter genes for real-time and quantitative analysis of messenger RNA silencing.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Luciferase genes have been used extensively for quantitative analysis in RNA interference (RNAi) and endogenous microRNA (miRNA) studies. However, one drawback is that determination of luciferase activity always requires that cells be killed, allowing less real-time information about a biological process to be obtained. Here we describe a triple-reporter plasmid for target miRNA analysis in which enhanced green fluorescent protein (EGFP) and Renilla luciferase (RLuci) are linked by "self-cleave" 2A under control of the CMV promoter. Firefly luciferase (FLuci) serves as internal control under control of another independ...
Source: Analytical Biochemistry - November 15, 2009 Category: Biochemistry Authors: Feng L, Li F, Liu Y, Zheng X, Zhang B, Chen L Tags: Anal Biochem Source Type: journals

Proteome analysis of a single zebrafish embryo using three different digestion strategies coupled with liquid chromatography-tandem mass spectrometry.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Zebrafish is a powerful model to analyze vertebrate embryogenesis and organ development. Although a number of genes have been identified to specify embryonic development processes, only a few large-scale proteomic analyses have been reported in regard to these events to date. Here the total proteins of a single embryo were analyzed by urea-, sodium deoxycholate (SDC)-, and performic acid (PA)-assisted trypsin digestion strategies coupled to capillary liquid chromatography-tandem mass spectrometry (CapLC-MS/MS) identification. In total, 509 and 210 proteins were detected from the embryos at 72 and 120 hours postfertiliz...
Source: Analytical Biochemistry - November 15, 2009 Category: Biochemistry Authors: Lin Y, Chen Y, Yang X, Xu D, Liang S Tags: Anal Biochem Source Type: journals

A gas chromatography-mass spectrometry method for the quantitation of N-nitrosoproline and N-acetyl-S-allylcysteine in human urine: application to a study of the effects of garlic consumption on nitrosation.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present a new method for extraction and sensitive detection of both NPRO and N-acetyl-S-allylcysteine from urine. The latter is a metabolite of S-allylcysteine, which is found in garlic. Urine was acidified and the organic acids were extracted by reversed-phase extraction (RP-SPE) and use of a polymeric weak anion exchange (WAX-SPE) resin. NPRO was quantified by isotope dilution gas chromatography-mass spectrometry (GC-MS) using [13C5]NPRO and N-nitrosopipecolinic acid (NPIC) as internal standards. This method was used to analyze urine samples from a study that was designed to test whether garlic supplementation inhibit...
Source: Analytical Biochemistry - November 15, 2009 Category: Biochemistry Authors: Cope K, Seifried H, Seifried R, Milner J, Kris-Etherton P, Harrison EH Tags: Anal Biochem Source Type: journals

An enzyme-coupled continuous spectrophotometric assay for magnesium protoporphyrin IX methyltransferases.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The second committed step in chlorophyll biosynthesis is the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to magnesium protoporphyrin IX (MgP) forming MgP monomethylester (MgPME). This reaction is catalyzed by the enzyme MgP methyltransferase (ChlM). Previous investigation of this enzyme has involved the use of time-consuming techniques requiring separation of products from substrates. More recent methyltransferase studies use coupling enzymes to monitor changes in absorption/fluorescence for the measurement of activity. However, due to the spectral properties of porphyrins, many of these assays are un...
Source: Analytical Biochemistry - November 15, 2009 Category: Biochemistry Authors: McLean S, Hunter CN Tags: Anal Biochem Source Type: journals

Transcription level of messenger RNA per gene copy determined with dual-spike-in strategy.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
To quantify the transcription level of a gene, we have conceived a novel concept, transcription level of messenger RNA (mRNA) per gene copy, which was determined with a dual-spike-in strategy. In this strategy, an exogenous DNA was added as the spike reference for target DNA in addition to the exogenous RNA as the reference for target RNA. After the mRNA-to-DNA ratio of a target gene was estimated by real-time polymerase chain reaction (PCR), it was first normalized with the mRNA-to-DNA ratio of the exogenous reference. The normalized ratio was multiplied by the ratio of exogenous RNA to exogenous DNA to obtain the tra...
Source: Analytical Biochemistry - November 15, 2009 Category: Biochemistry Authors: Zhang Y, Wei Z, Li YY, Chen Y, Shen W, Lu C Tags: Anal Biochem Source Type: journals

A specific colorimetric assay for measuring transglutaminase 1 and factor XIII activities.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we use these substrates in specific tests for measuring the activities of TGase 1 and factor XIII. PMID: 19646949 [PubMed - indexed for MEDLINE] (Source: Analytical Biochemistry)
Source: Analytical Biochemistry - November 15, 2009 Category: Biochemistry Authors: Hitomi K, Kitamura M, Alea MP, Ceylan I, Thomas V, El Alaoui S Tags: Anal Biochem Source Type: journals

An introduction to methods for analyzing thiols and disulfides: Reactions, reagents, and practical considerations.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
PMID: 19664585 [PubMed - indexed for MEDLINE] (Source: Analytical Biochemistry)
Source: Analytical Biochemistry - November 15, 2009 Category: Biochemistry Authors: Hansen RE, Winther JR Tags: Anal Biochem Source Type: journals

Identification of farnesoid X receptor modulators by a fluorescence polarization-based interaction assay.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Farnesoid X receptor (FXR) serves as a receptor for chenodeoxycholic acid (CDCA) and other bile acids, and coordinates cholesterol and lipid metabolism. Since targeting the FXR-CDCA interaction thus might provide a way to regulate lipid homeostasis, we developed an FXR binding assay based on fluorescence polarization. Employing a fluorescently-labeled CDCA-F, we showed that CDCA-F selectively bound to the ligand binding domain of FXR (FXR-LBD) among nuclear receptors. The assay was then utilized for screening inhibitors against the FXR-CDCA interaction, thereby discovering four relatively potent inhibitors. The selecte...
Source: Analytical Biochemistry - November 11, 2009 Category: Biochemistry Authors: Han KC, Kim JH, Kim KH, Kim EE, Seo JH, Yang EG Tags: Anal Biochem Source Type: journals

Identification of RNA Linkage Isomers by Anion-Exchange Purification with ESI-MS of Automatically-Desalted Phosphodiesterase-II Digests.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Among the possible contaminants unique to RNA are linkage isomers that are difficult to identify by standard oligonucleotide analysis techniques. In a prior report we used non-porous and monolithic polymer anion exchangers for purification, and demonstrated a method to identify the presence of the linkage isomers. We also suggested a confirming technique employing phosphodiesterase-II (PDase-II), an enzyme incapable of cleaving 2 -5 linkages. We now present a method identifying the location of the linkage in the RNA isomer by anion-exchange purification and ESI-MS of the digestion products. Because the IP-RPLC desaltin...
Source: Analytical Biochemistry - November 11, 2009 Category: Biochemistry Authors: Thayer JR, Puri N, Burnett C, Hail M, Rao S Tags: Anal Biochem Source Type: journals

Development of inductively coupled plasma-mass spectrometry (ICP-MS) based protease assays.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Rapid, sensitive and quantitative assays for proteases are important for drug development and in the diagnosis of disease. Here, an assay for protease activity which uses inductively coupled plasma-mass spectrometry (ICP-MS) detection is described. Peptidic alpha-chymotrypsin substrates were synthesized containing a lanthanide ion chelate at the N-terminus to provide a distinct elemental tag. A biotin label was appended to the C-terminus of the peptide allowing separation of uncleaved peptide from the enzymatic digestion. The enzyme activity was determined by quantifying the lanthanide ion signal of the peptide cleavag...
Source: Analytical Biochemistry - November 10, 2009 Category: Biochemistry Authors: Lathia US, Ornatsky O, Baranov V, Nitz M Tags: Anal Biochem Source Type: journals

Quantitation of Folates and their Catabolites in Blood Plasma, Erythrocytes and Urine by Stable Isotope Dilution Assays.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
New stable isotope dilution assays were developed for the simultaneous quantitation of the folates 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, tetrahydrofolic acid, 10-formylfolic acid and folic acid as well as for their catabolites para-aminobenzoylglutamate (pABG) and acetyl-para-aminobenzoylglutamate (ApABG) in clinical samples. The methods were based on clean up by strong anion exchange followed by LC-MS/MS detection. Deuterated analogues of the folates and [(13)C(5)]-labelled isotopologues of the catabolites were applied as the internal standards in stable isotope dilution assays. Extraction in 4-m...
Source: Analytical Biochemistry - November 7, 2009 Category: Biochemistry Authors: Mönch S, Netzel M, Netzel G, Rychlik M Tags: Anal Biochem Source Type: journals

Selection of thiol- and disulfide-containing proteins of Escherichia coli on activated thiol sepharose.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Activated thiol sepharose (ATS) facilitates selection of thiol-containing proteins. In control- and menadione-treated Escherichia coli, batch selection performed under denaturing conditions revealed distinct two dimensional electrophoresis (2DE) patterns. Using shotgun proteomics, 183 thiol-containing proteins were identified in control ATS-selected extracts, 126 in menadione-treated E. coli with 85 proteins common to both. More than 90% of identified proteins contained one or more cysteines. Blocking with N-ethyl maleimide followed by reduction facilitated ATS-based selection of disulfide-containing proteins. 62 prote...
Source: Analytical Biochemistry - November 7, 2009 Category: Biochemistry Authors: Hu W, Tedesco S, McDonagh B, Bárcena JA, Keane C, Sheehan D Tags: Anal Biochem Source Type: journals

Facile Construction of Fluorescent Peptide Microarrays: One-Step Fluorescent Derivatization of Sub-microscale Peptide Aldehydes for Selective Terminal Immobilization.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this note, we demonstrate the utility of bifunctional fluorescent linkers to facilitate the construction of peptide microarrays with either an N or C-terminal alkylamine for directionally preferred peptide immobilization. Significantly, these small tags facilitate HPLC profiling, while limiting interference with antigen-antibody interactions after peptide immobilization. In a model peptide-antibody binding assay, a sequence-dependent orientation effect of antibody binding to a series of peptide ligands was demonstrated. This approach provides a strategy that can be applied to a variety of peptide-microarray based de...
Source: Analytical Biochemistry - November 7, 2009 Category: Biochemistry Authors: Dong H, Song X, Lasanajak Y, Cummings RD, Chaikof EL Tags: Anal Biochem Source Type: journals

Template-blocking PCR: An advanced PCR technique for genome walking.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This report describes the development of an improved method for the isolation of genomic fragments adjacent to a known DNA sequence based on a cassette-ligation-mediated PCR technique. To reduce the nonspecific amplification of PCR-based genome walking, the 3' ends of the restriction enzyme digested genomic DNA fragments were blocked with dideoxy NTP (ddNTP) and ligated with properly designed cassettes. The modified genomic DNA fragments flanked with cassettes were used as a template for the amplification of a target gene with a gene-specific primer (GSP) and cassette primer. The ddNTP blocking of the genomic DNA ends sign...
Source: Analytical Biochemistry - November 7, 2009 Category: Biochemistry Authors: Bae JH, Sohn JH Tags: Anal Biochem Source Type: journals

Bacteria Capture, Lysate Clearance, and Plasmid DNA Extraction Using pH-Sensitive Multifunctional Magnetic Nanoparticles.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A multifunctional magnetic nanoparticle (MNP) assisted bio-separation method was developed to isolate plasmid DNA (pDNA) from Escherichia coli culture. Using the pH-sensitive carboxyl-modified magnetic nanoparticles, both cell capture and the subsequent removal of genomic-DNA/protein complex after lysis can be simply achieved by magnetic separation. Further, the yield and purity of pDNA extracted by MNPs is comparable to that obtained using organic solvents or commercial kits. This time and cost-effective protocol does not require centrifugation or precipitation steps, and has the potential for automated DNA extraction...
Source: Analytical Biochemistry - November 7, 2009 Category: Biochemistry Authors: Shan Z, Wu Q, Wang X, Zhou Z, Oakes KD, Zhang X, Huang Q, Yang W Tags: Anal Biochem Source Type: journals

7-Benzyloxyresorufin-O-dealkylase activity as a marker for measuring cytochrome P450 CYP3A induction in mouse liver.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The cytochrome P450 subfamily CYP3A belongs to the most important detoxification enzymes. As the main CYP3A isoforms are not polymorphic and therefore detract themselves from genetic screening as a potent prediction marker for drug metabolism or induction effects, effective in vitro testing of a putative drug - CYP3A interaction is indicated. We used mouse liver microsomes treated with the model drug phenytoin to set up an effective and reliable in vitro test system. A metabolic assay analyzing 7-alkoxyresorufin-O-dealkylation showed specific CYP3A-dependent 7-benzyloxyresorufin oxidation (BROD). This was confirmed by ...
Source: Analytical Biochemistry - November 7, 2009 Category: Biochemistry Authors: Hagemeyer CE, Bürck C, Schwab R, Knoth R, Meyer RP Tags: Anal Biochem Source Type: journals

High-resolution melting analysis for genotyping of the myotonic dystrophy type 1 associated Alu insertion/deletion polymorphism.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Since its introduction, high-resolution melting (HRM) analysis has been used for genotyping of various types of sequence alterations. In our study we report the utilization of HRM for genotyping of the 1kb insertion/deletion polymorphism, involving a problematic region of five consecutive Alu elements, which is associated with myotonic dystrophy type 1. We combined a three-primer PCR amplification approach with HRM using two primer sets. Analyses based on curve shapes are sensitive enough to differentiate between genotypes with both primer sets. In addition, the newly designed insertion specific primer from the second ...
Source: Analytical Biochemistry - November 7, 2009 Category: Biochemistry Authors: Radvansky J, Resko P, Surovy M, Minarik G, Ficek A, Kadasi L Tags: Anal Biochem Source Type: journals

Novel fluorescent polarization-based assay for the identification of disruptors of the RCAN1/calcineurin A protein complex.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We report here the development of an optimized in vitro high-throughput fluorescence polarization assay based on the disruption of the RCAN1(198-218)-CnA interaction for identifying molecules with immunosuppressant potential. This approach led us to identify dipyridamole as a disruptor of such interaction whose immunosuppressive effect was confirmed in cellular models. Moreover, three small molecules with a potential immunosuppressive effect were also identified. PMID: 19891949 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry)
Source: Analytical Biochemistry - November 2, 2009 Category: Biochemistry Authors: Mulero MC, Orzáez M, Messeguer J, Messeguer A, Pérez-Payá E, Pérez-Riba M Tags: Anal Biochem Source Type: journals

Point spread function effect in image-based fluorescent microplate detection.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A major advantage of the image based fluorescent microplate detection approach is the capability of using lower magnification to maximize the field of view so that color or intensity can provide first steps to isolating samples of interest in a high throughput fashion. Lenses with low magnification typically have low numerical aperture, consequently the effect of the point spread function and liquid meniscus effect of small liquid volumes on standard microplates conspire to affect the intensity readings. We demonstrate this and show that the capillary wells microplate design overcomes this limitation. (c) 2009 Elsevier...
Source: Analytical Biochemistry - November 2, 2009 Category: Biochemistry Authors: Tan HY, Ng TW, Neild A, Liew OW Tags: Anal Biochem Source Type: journals

OBOC Peptide Library Sequencing via High-Pressure Ammonia Cleavage Coupled with Nanomanipulation/Nanoelectrospray Mass Spectrometry.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Biological screening of one-bead, one-compound (OBOC) combinatorial peptide libraries is routinely carried out with the peptide remaining bound to the resin bead during screening. After a hit is identified, the bead is isolated, the peptide cleaved from the bead, and its sequence determined. We have developed a new technique for cleavage of peptides from resin beads whereby exposure of an HMBA-linked peptide to high pressure ammonia gas led to efficient cleavage in as little as 5 min. Here we also report a new method of extracting peptide from individual library beads for its introduction into a mass spectrometer that ...
Source: Analytical Biochemistry - November 2, 2009 Category: Biochemistry Authors: Brown JM, Hoffmann WD, Alvey CM, Wood AR, Verbeck GF, Petros RA Tags: Anal Biochem Source Type: journals

A novel SNP detection of a double-stranded DNA target by a ribonucleotide-carrying molecular beacon and thermostable RNase HII.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this report, we describe a novel method for SNP genotyping, based on differential fluorescence emission due to cleavage by Thermus thermophilus RNase HII (TthRNase HII) of DNA heteroduplexes containing a SNP-site-specific chimeric DNA-rN(1)-DNA molecular beacon (cMB). We constructed a loop sequence for a cMB that contains a single SNP-specific ribonucleotide at the central site. When the cMB probe is hybridized to a target dsDNA, a perfect match of the cMB/DNA duplex permits efficient cleavage with TthRNase HII, whereas a mismatch in the duplex due to a SNP greatly reduces efficiency. Cleavage efficiency is measured by ...
Source: Analytical Biochemistry - November 2, 2009 Category: Biochemistry Authors: Liu XP, Hou JL, Liu JH Tags: Anal Biochem Source Type: journals

Enrichment of serum low molecular weight proteins using C(18) absorbent under urea/DTT denatured environment.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Serum low molecular weight proteins (LMWPs, molecular weight of less than 30 kDa) are closely related with the body physiological and pathological situations, while many difficulties encountered when enriching and fractionating them. Using C(18) absorbent (100 A) enrichment and fractionation under urea/DTT denatured environment, followed by 60% ACN elution, serum LMWPs could be enriched more than 100 folds, which were evaluated by SDS-PAGE, 2-DE, and ICAT labeling quantification. Proteins existed in human serum at low ng/ml level such as MRP proteins could be identified directly from 2-DE coupled with MALDI TOF/TOF MS ...
Source: Analytical Biochemistry - November 2, 2009 Category: Biochemistry Authors: Wu J, An Y, Pu H, Shan Y, Ren X, An M, Wang Q, Wei S, Ji J Tags: Anal Biochem Source Type: journals

Disposable bioluminescence-based biosensor for detection of bacterial count in food.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A biosensor for rapid detection of bacterial count based on adenosine 5'-triphosphate (ATP) bioluminescence has been developed. The biosensor is composed of a key sensitive element and a photomultiplier tube used as a detector element. The disposable sensitive element consists of a sampler, a cartridge where intracellular ATP is chemically extracted from bacteria, and a microtube where the extracted ATP reacts with the luciferin-luciferase reagent to produce bioluminescence. The bioluminescence signal is transformed into relevant electrical signal by the detector and further measured with a homemade luminometer. Parame...
Source: Analytical Biochemistry - November 1, 2009 Category: Biochemistry Authors: Luo J, Liu X, Tian Q, Yue W, Zeng J, Chen G, Cai X Tags: Anal Biochem Source Type: journals

Effect of storage regime on the stability of DNA used as a calibration standard for real-time polymerase chain reaction.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This article looks at storage factors influencing the stability of potential DNA calibration standards for use in quantitative polymerase chain reaction (PCR). Target sequences from the bacteria Campylobacter jejuni were cloned into a plasmid vector. Samples of these potential calibration standards were stored at +4, -20, and -80 degrees C as aqueous and lyophilized samples and were prepared as both single-use aliquots and multiple-use preparations. Results showed that the samples stored as single-use aqueous solutions at +4 degrees C and lyophilized samples stored at +4 and -20 degrees C were the most stable. Samples stor...
Source: Analytical Biochemistry - November 1, 2009 Category: Biochemistry Authors: Podivinsky E, Love JL, van der Colff L, Samuel L Tags: Anal Biochem Source Type: journals

Identification of heme propionate vibrational modes in the resonance Raman spectra of cytochrome c oxidase.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The propionate groups of heme a and a(3) in cytochrome c oxidase (CcO) have been postulated to mediate both the electron and proton transfer within the enzyme. To establish structural markers for the propionate groups, their associated vibrational modes were identified in the resonance Raman spectra of CcO from bovine (bCcO) and Rhodobacter sphaeroides (RsCcO). The distinction between the modes from the propionates of heme a and heme a(3), as well as those from the propionates on the pyrrole rings A and D in each heme, was made on the basis of H2O-D2O isotope substitution experiments combined with wavelength-selective ...
Source: Analytical Biochemistry - November 1, 2009 Category: Biochemistry Authors: Egawa T, Lee HJ, Ji H, Gennis RB, Yeh SR, Rousseau DL Tags: Anal Biochem Source Type: journals

A homogeneous resonance energy transfer assay for phosphopantetheinyl transferase.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Phosphopantetheinyl transferase plays an essential role in activating fatty acid, polyketide, and nonribosomal peptide biosynthetic pathways, catalyzing covalent attachment of a 4'-phosphopantetheinyl group to a conserved residue within carrier protein domains. This enzyme has been validated as an essential gene to primary metabolism and presents a target for the identification of antibiotics with a new mode of action. Here we report the development of a homogeneous resonance energy transfer assay using fluorescent coenzyme A derivatives and a surrogate peptide substrate that can serve to identify inhibitors of this en...
Source: Analytical Biochemistry - November 1, 2009 Category: Biochemistry Authors: Foley TL, Burkart MD Tags: Anal Biochem Source Type: journals

Identification of Nile red as a fluorescent substrate of the Candida albicans ATP-binding cassette transporters Cdr1p and Cdr2p and the major facilitator superfamily transporter Mdr1p.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Clinically relevant azole resistance in the fungal pathogen Candida albicans is most often associated with the increased expression of plasma membrane efflux pumps, specifically the ATP-binding cassette (ABC) transporters CaCdr1p and CaCdr2p and the major facilitator superfamily (MFS) transporter CaMdr1p. Development of potent pump inhibitors that chemosensitize cells to azoles is a promising approach to overcome antifungal resistance. Here we identify Nile red as a new fluorescent substrate for CaCdr1p, CaCdr2p, and CaMdr1p. Nile red was effluxed efficiently from Saccharomyces cerevisiae cells heterologously expressin...
Source: Analytical Biochemistry - November 1, 2009 Category: Biochemistry Authors: Ivnitski-Steele I, Holmes AR, Lamping E, Monk BC, Cannon RD, Sklar LA Tags: Anal Biochem Source Type: journals

Differential binding of tropomyosin isoforms to actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester and fluorescein-5-isothiocyanate.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Differential interactions of tropomyosin (TM) isoforms with actin can be important for determination of the thin filament functions. A mechanism of tropomyosin binding to actin was studied by comparing interactions of five alphaTM isoforms with actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and with fluorescein-5-isothiocyanate (FITC). MBS attachment sites were revealed with mass spectrometry methods. We found that the predominant actin fraction was cross-linked by MBS within subdomain 3. A smaller fraction of the modified actin was cross-linked within subdomain 2 and between subdomains 2 and 1...
Source: Analytical Biochemistry - November 1, 2009 Category: Biochemistry Authors: Skórzewski R, Robaszkiewicz K, Jarzebińska J, Suder P, Silberring J, Moraczewska J Tags: Anal Biochem Source Type: journals

A new homogeneous high-throughput screening assay for profiling compound activity on the human ether-a-go-go-related gene channel.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Long QT syndrome, either inherited or acquired from drug treatments, can result in ventricular arrhythmia (torsade de pointes) and sudden death. Human ether-a-go-go-related gene (hERG) channel inhibition by drugs is now recognized as a common reason for the acquired form of long QT syndrome. It has been reported that more than 100 known drugs inhibit the activity of the hERG channel. Since 1997, several drugs have been withdrawn from the market due to the long QT syndrome caused by hERG inhibition. Food and Drug Administration regulations now require safety data on hERG channels for investigative new drug (IND) applica...
Source: Analytical Biochemistry - November 1, 2009 Category: Biochemistry Authors: Titus SA, Beacham D, Shahane SA, Southall N, Xia M, Huang R, Hooten E, Zhao Y, Shou L, Austin CP, Zheng W Tags: Anal Biochem Source Type: journals

Characterization of cluster-assembled nanostructured titanium oxide coatings as substrates for protein arrays.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Protein microarray technologies are rapidly expanding to fulfill current needs of proteome discovery for disease management. Nanostructured materials have been shown to present interesting features when used in biological settings: nanostructured titanium oxide film (ns-TiOx), synthesized by supersonic cluster beam deposition (SCBD), has recently emerged as a biocompatible substrate in different biological assays. The ns-TiOx surface is characterized by a morphology at the nanoscale that can be tuned to modulate specific biomolecule-material interactions. Here we present a systematic characterization of ns-TiOx coating...
Source: Analytical Biochemistry - November 1, 2009 Category: Biochemistry Authors: Carbone R, De Marni M, Zanardi A, Vinati S, Barborini E, Fornasari L, Milani P Tags: Anal Biochem Source Type: journals

Development of a charcoal paper adenosine triphosphate:pyrophosphate exchange assay: kinetic characterization of NEDD8 activating enzyme.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Ubiquitin activating enzyme (UAE, UBE1, or E1) and seven known homologous "E1s" initiate the conjugation pathways for ubiquitin and 16 other ubiquitin-like modifiers (ULMs) found in humans. The initial step catalyzed by E1s uses adenosine triphosphate (ATP) to adenylate the C terminus of the appropriate ULM and results in the production of inorganic pyrophosphate (PPi). The mechanism of these enzymes can be studied with assays that measure the rate of ULM-dependent ATP:PPi exchange. The traditional method follows the initial velocity of [32P]PPi incorporation into ATP by capturing the nucleotide on activated charcoal p...
Source: Analytical Biochemistry - November 1, 2009 Category: Biochemistry Authors: Bruzzese FJ, Tsu CA, Ma J, Loke HK, Wu D, Li Z, Tayber O, Dick LR Tags: Anal Biochem Source Type: journals

A continuous microplate assay for sirtuins and nicotinamide-producing enzymes.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases (sirtuins) and other enzymes that produce nicotinamide are integral to many cellular processes. Yet current activity measurements involve expensive and time-consuming assays. Here we present a spectroscopic assay that circumvents many issues of previous methods. This assay permits continuous product monitoring over time, allows determination of steady-state kinetic parameters, and is readily adaptable to high-throughput screening. The methodology uses an enzyme-coupled system in which nicotinamide is converted to nicotinic acid and ammonia by nicot...
Source: Analytical Biochemistry - November 1, 2009 Category: Biochemistry Authors: Smith BC, Hallows WC, Denu JM Tags: Anal Biochem Source Type: journals

Identification of suitable reference genes for gene expression studies of human serous ovarian cancer by real-time polymerase chain reaction.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, 10 housekeeping genes, ACTB, ALAS1, GAPDH, GUSB, HPRT1, PBGD, PPIA, PUM1, RPL29, and TBP as well as 18S rRNA that were already used in various studies were analyzed to determine their applicability. Totally 20 serous ovarian cancer specimens and 20 normal ovarian epithelial tissue specimens were examined. All candidate reference genes showed significant differences in expression between malignant and nonmalignant groups except GUSB, PPIA, and TBP. The expression stability and suitability of the 11 genes were validated employing geNorm and NormFinder. GUSB, PPIA, and TBP were demonstrated as the most stable r...
Source: Analytical Biochemistry - November 1, 2009 Category: Biochemistry Authors: Li YL, Ye F, Hu Y, Lu WG, Xie X Tags: Anal Biochem Source Type: journals

Coexpression of CYP11B2 or CYP11B1 with adrenodoxin and adrenodoxin reductase for assessing the potency and selectivity of aldosterone synthase inhibitors.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Excessive production of aldosterone has been implicated in the pathogenesis of hypertension and heart failure. One approach to ameliorate the deleterious effects of aldosterone is to suppress its biosynthesis. The enzyme aldosterone synthase (CYP11B2) is responsible for the final step of aldosterone synthesis. It requires electron transfer from the adrenodoxin/adrenodoxin reductase system to catalyze the production of aldosterone. A stable cell line simultaneously overexpressing recombinant human CYP11B2 as well as human adrenodoxin and adrenodoxin reductase was established to help maximize the enzyme activity. The hom...
Source: Analytical Biochemistry - November 1, 2009 Category: Biochemistry Authors: LaSala D, Shibanaka Y, Jeng AY Tags: Anal Biochem Source Type: journals

Differentiation of isomeric Lewis blood groups by positive ion electrospray tandem mass spectrometry.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Lewis histo-blood group antigens are one of the major classes of biologically active oligosaccharides. In this work, underivatized Lewis blood groups were studied by electrospray mass spectrometry (ESI-MS(n)) in the positive mode with three different mass analysers (Q-TOF, QqQ, and LIT). It was observed that, under collision induced fragmentations, Type 1 Lewis antigens (Le(a) and Le(b)) could be distinguished from Type 2 (Le(x) and Le(y)) on the basis of specific fragmentations of the GlcNAc unit. While O-4 linked sugars of the GlcNAc are lost as residues, the O-3 linked sugars undergo fragmentation both as sugar unit...
Source: Analytical Biochemistry - October 27, 2009 Category: Biochemistry Authors: Ferreira JA, Domingues MR, Reis A, Monteiro MA, Coimbra MA Tags: Anal Biochem Source Type: journals

Improving discrimination of outer membrane proteins by fusing different forms of pseudo amino acid composition.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, some sequence-derived structural and physicochemical features of proteins are incorporated with amino acid composition to discriminate OMPs from non-OMPs using support vector machines. The discrimination performance of the proposed method is evaluated on a benchmark dataset of 208 OMPs, 673 globular proteins and 206 alpha helical membrane proteins. A high overall accuracy of 97.8% was observed in the 5-fold cross-validation test. Additionally, the present method distinguished OMPs from globular proteins and alpha helical membrane proteins with an overall accuracy of 98.2% and 96.4%, respectively. The predict...
Source: Analytical Biochemistry - October 26, 2009 Category: Biochemistry Authors: Gao QB, Ye XF, Jin ZC, He J Tags: Anal Biochem Source Type: journals

A spectrophotometric method for the determination of zinc, copper and cobalt ions in metalloproteins using Zincon.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Zincon (2-carboxy-2'hydroxy-5'sulfoformazylbenzene) has long been known as an excellent colorimetric reagent for the detection of zinc and copper ions in aqueous solution. To extend the chelator's versatility to the quantification of metal ions in metalloproteins, the spectral properties of Zincon and its complexes with Zn(2+), Cu(2+) and Co(2+) were investigated in the presence of guanidine hydrochloride and urea, two common denaturants used to labilize metal ions in proteins. These studies revealed the detection of metals to be generally more sensitive with urea. In addition, pH profiles recorded for these metals ind...
Source: Analytical Biochemistry - October 22, 2009 Category: Biochemistry Authors: Säbel CE, Neureuther JM, Siemann S Tags: Anal Biochem Source Type: journals

TD-DFT-assisted absolute configuration determination of cis-dihydrodiol metabolite produced from isoflavone by biphenyl dioxygenase.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Escherichia coli cells containing the biphenyl dioxygenase genes bphA1A2A3A4 from Pseudomonas pseudoalcaligenes KF707 were found to biotransform isoflavone and produced a metabolite that was not found in a control experiment. LC/MS and (1)H and (13)C-NMR analyses indicated biphenyl dioxygenase induced 2',3'-cis-dihydroxylation of the B-ring of isoflavone. In a previous report, the same enzyme showed dioxygenase activity towards flavone, producing flavone 2',3'-cis-dihydrodiol. Due to growing interest in flavone chemistry and the absolute configuration of natural products, TD-DFT calculations were combined with CD spect...
Source: Analytical Biochemistry - October 22, 2009 Category: Biochemistry Authors: Seo J, Kang SI, Kim M, Won D, Takahashi H, Ahn JH, Chong Y, Lee E, Lim Y, Kanaly RA, Han J, Hur HG Tags: Anal Biochem Source Type: journals

A Continuous Assay for alpha-Methylacyl-CoA Racemase (AMACR) Using Circular Dichroism.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
a-Methylacyl-CoA racemase (AMACR) catalyzes the epimerization of (2R)- and (2S)-methyl branched fatty acyl coenzyme A-thioesters. AMACR is a biomarker for prostate cancer and a putative target for the development of therapeutic agents directed against the disease. To facilitate development of AMACR inhibitors, a continuous CD-based assay has been developed. The open reading frame encoding AMACR from Mycobacterium tuberculosis (MCR) was sub-cloned into a pET-15b vector and the enzyme was overexpressed and purified using metal ion affinity chromatography. The rates of MCR-catalyzed epimerization of either (2R)- or (2S)-i...
Source: Analytical Biochemistry - October 22, 2009 Category: Biochemistry Authors: Ouazia D, Bearne SL Tags: Anal Biochem Source Type: journals

Comparative determination of some phytohormones in wild-type and genetically modified plants by gc-ms and hplc-ms/ms.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The analytical performances of two optimized analytical methodologies used for the determination of auxins, cytokinins and abscisic acid in plant samples were critically compared. Phytohormones were extracted from Nicotiana glauca samples using a modified Bieleski solvent and determined both by gas chromatography-mass spectrometry (GC-MS), after derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and by high performance liquid chromatography tandem mass spectrometry-mass spectrometry (HPLC-MS/MS) on the Bieleski extract without any further treatment. HPLC-MS/MS gave better results in terms of higher ...
Source: Analytical Biochemistry - October 22, 2009 Category: Biochemistry Authors: Giannarelli S, Muscatello B, Bogani P, Spiriti MM, Buiatti M, Fuoco R Tags: Anal Biochem Source Type: journals

A robust assay measuring GLUT4 translocation in rat myoblasts overexpressing Glut4myc and AS160_v2.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Muscle and fat cells translocate the glucose transporter 4, GLUT4, to the plasma membrane when stimulated by insulin. Usually this event is measured in differentiated adipocytes, myotubes, or cell lines overexpressing tagged GLUT4, by immunostaining. However, measurement of the translocation in differentiated adipocytes or myotubes or GLUT-4 overexpressing cell lines is difficult because of high assay variability caused by either the differentiation protocol or low assay sensitivity. We recently reported the identification of a novel splice variant of AS160 (AS160_v2) and showed that its co-expression with GLUT4 in L6 ...
Source: Analytical Biochemistry - October 22, 2009 Category: Biochemistry Authors: Baus D, Yan Y, Li Z, Garyantes T, Hoop MD, Tennagels N Tags: Anal Biochem Source Type: journals

Mammalian Oxa1 protein is useful for assessment of submitochondrial protein localization and mitochondrial membrane integrity.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Taking advantage of the unique topology of Oxa1 protein, a mitochondrial inner membrane protein with N (inter-membrane space)-C (matrix) orientation, we explored the usefulness of the protein as a marker for submitochondrial protein localization. Mammalian Oxa1 protein exhibited different proteolytic patterns depending on mitochondrial membrane integrity and, in mitochondria with a disrupted outer membrane and outer and inner membranes, the proteolytic patterns of Oxa1 protein were consistent with those of mitochondrial inter-membrane space and matrix marker proteins, respectively, suggesting that Oxa1 protein, a singl...
Source: Analytical Biochemistry - October 22, 2009 Category: Biochemistry Authors: Sato T, Mihara K Tags: Anal Biochem Source Type: journals

Vibrational Spectroscopy and Chemometrics to Characterize and Quantitate Trehalose Crystallization.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, Raman and Near Infrared (NIR) Spectroscopy are used to characterize and quantitate trehalose crystallization using several chemometric models. The predictive behavior of partial least squares (PLS), principal component analysis (PCA) and multiple linear regression (MLR) models are compared. In general, PLS and PCA outperform linear and MLR models. Changes in specific vibrational modes associated with several coupled motions are described and assigned as a function of crystal content. In addition to characterization and quantitation, our method may be used to localize gradients of amorphous and/or crystallize...
Source: Analytical Biochemistry - October 21, 2009 Category: Biochemistry Authors: Connolly B, Patapoff TW, Wang YJ, Moore JM, Kamerzell TJ Tags: Anal Biochem Source Type: journals

Optimization of Time-Resolved Fluorescence Assay for Detection of Eu-DOTA-labeled Ligand-Receptor Interactions.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Lanthanide-based luminescent ligand binding assays are superior to traditional radiolabel assays due to improved sensitivity and affordability in high throughput screening while eliminating the use of radioactivity. Despite significant progress using lanthanide(III)-coordinated chelators such as DTPA derivatives, dissociation-enhanced lanthanide fluoroimmunoassays (DELFIA) have not yet been successfully used with more stable chelators, e.g. DOTA derivatives, due to the incomplete release of lanthanide(III) ions from the complex. Here, a modified and an optimized DELFIA procedure incorporating an acid treatment protocol...
Source: Analytical Biochemistry - October 20, 2009 Category: Biochemistry Authors: Silva CR, Vagner J, Lynch R, Gillies RJ, Hruby VJ Tags: Anal Biochem Source Type: journals

Influences of electrolytes and glucose on formulation of carbonate apatite nano-crystals for efficient gene delivery to mammalian cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Genetic manipulation of human cells through delivery of a functional gene or a gene-silencing element is an attractive approach to treat critical diseases very precisely and effectively. Extensive research on the genetic basis of human diseases with complete sequencing of human genome revealed many vital genes as possible targets in gene therapy programs. On the other hand, to facilitate cell or tissue-directed delivery of genes and gene-silencing nucleic acid sequences, both genetic and chemical engineering approaches led to the generation of various viral and non-viral carriers. However, considering the issues of bot...
Source: Analytical Biochemistry - October 20, 2009 Category: Biochemistry Authors: Hossain S, Tada S, Akaike T, Chowdhury EH Tags: Anal Biochem Source Type: journals