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157 records returned

Fluorescent oligonucleotides can serve as suitable alternatives to radiolabeled oligonucleotides.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The objective of this study was to find an alternative to the use of radioactivity in medical research using nucleic acid chemistry. FITC-labeled oligonucleotides that contain wild-type (wt) and modified base (8-oxo-G) at the same position and their complementary unlabeled strand were synthesized. Purified DNA repair enzyme, OGG1, and nuclear lysates from MCF-7 breast cancer cells were incubated with double-stranded FITC-labeled wt and 8-oxo-G oligonucleotide to demonstrate the OGG1 incision assay. We found that FITC-coupled oligonucleotides do not impose a steric hindrance during duplex formation, and the fluorescence int...
Source: Journal of Biomolecular Techniques : JBT - August 31, 2009 Category: Molecular Biology Authors: Ballal R, Cheema A, Ahmad W, Rosen EM, Saha T Tags: J Biomol Tech Source Type: journals

Generation of human PTH1R construct with flag epitope located internally: comparison of two-fragment assembly by using PCR overlap extension or ligase.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Parathyroid hormone (PTH) regulates bone remodeling and calcium and phosphate homeostasis. PTH actions are mediated by type I PTH/PTH-related peptide receptor (PTH1R). There has been no commercially available, specific antibody to detect human PTH1R expression so far. Flag-tagged human PTH1R construct, converting the sequence DKEAPTGS (residues 94-101) in the exon E2 region of PTH1R to DYKDDDDK of Flag epitope, was generated by using PCR overlap extension or ligase enzyme for two-fragment assembly. We found that Flag-tagged PTH1R assembled by ligase was easy to be manipulated, but its efficiency was lower than that of ...
Source: Journal of Biomolecular Techniques : JBT - August 31, 2009 Category: Molecular Biology Authors: Wang B, Yang Y Tags: J Biomol Tech Source Type: journals

A comparison of protein extraction methods suitable for gel-based proteomic studies of aphid proteins.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Protein extraction methods can vary widely in reproducibility and in representation of the total proteome, yet there are limited data comparing protein isolation methods. The methodical comparison of protein isolation methods is the first critical step for proteomic studies. To address this, we compared three methods for isolation, purification, and solubilization of insect proteins. The aphid Schizaphis graminum, an agricultural pest, was the source of insect tissue. Proteins were extracted using TCA in acetone (TCA-acetone), phenol, or multi-detergents in a chaotrope solution. Extracted proteins were solubilized in a...
Source: Journal of Biomolecular Techniques : JBT - August 31, 2009 Category: Molecular Biology Authors: Cilia M, Fish T, Yang X, McLaughlin M, Thannhauser TW, Gray S Tags: J Biomol Tech Source Type: journals

The ABRF Edman Sequencing Research Group 2008 Study: investigation into homopolymeric amino acid N-terminal sequence tags and their effects on automated Edman degradation.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and executes interlaboratory studies investigating the use of automated Edman degradation for protein and peptide analysis. In 2008, the ESRG enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. Commonly, to facilitate protein purification, an affinity tag containing a polyhistidine sequence is conjugated to the N-terminus of the protein. After expression, polyhistidine-tagged protein is readily purified via chelation with an immobilized metal a...
Source: Journal of Biomolecular Techniques : JBT - August 31, 2009 Category: Molecular Biology Authors: Thoma RS, Smith JS, Sandoval W, Leone JW, Hunziker P, Hampton B, Linse KD, Denslow ND Tags: J Biomol Tech Source Type: journals

A framework for managing core facilities within the research enterprise.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Core facilities represent increasingly important operational and strategic components of institutions' research enterprises, especially in biomolecular science and engineering disciplines. With this realization, many research institutions are placing more attention on effectively managing core facilities within the research enterprise. A framework is presented for organizing the questions, challenges, and opportunities facing core facilities and the academic units and institutions in which they operate. This framework is intended to assist in guiding core facility management discussions in the context of a portfolio of...
Source: Journal of Biomolecular Techniques : JBT - August 31, 2009 Category: Molecular Biology Authors: Haley R Tags: J Biomol Tech Source Type: journals

Multiplex amplicon genotyping by high-resolution melting.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this report, a single instrument that combines rapid-cycle real-time PCR with high-resolution melting [LightScanner-32 (LS-32), Idaho Technology, Salt Lake City, UT] was used for multiplex amplicon genotyping. The four most common mutations associated with thrombophilia, F5 (factor V Leiden 1691G>A), F2 (prothrombin 20210G>A), and methylenetetrahydrofolate reductase (MTHFR; 1298A>C and 677C>T) were genotyped in a single homogeneous assay with internal controls to adjust for minor chemistry and instrument variation. Forty temperature cycles required 9.2 min, and each capillary required 2.2 min by melting at 0...
Source: Journal of Biomolecular Techniques : JBT - June 30, 2009 Category: Molecular Biology Authors: Seipp MT, Durtschi JD, Voelkerding KV, Wittwer CT Tags: J Biomol Tech Source Type: journals

A Method for Preparing DNA Sequencing Templates Using a DNA-Binding Microplate.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A DNA-binding matrix was immobilized on the surface of a 96-well microplate and used for plasmid DNA preparation for DNA sequencing. The same DNA-binding plate was used for bacterial growth, cell lysis, DNA purification, and storage. In a single step using one buffer, bacterial cells were lysed by enzymes, and released DNA was captured on the plate simultaneously. After two wash steps, DNA was eluted and stored in the same plate. Inclusion of phosphates in the culture medium was found to enhance the yield of plasmid significantly. Purified DNA samples were used successfully in DNA sequencing with high consistency and r...
Source: Journal of Biomolecular Techniques : JBT - June 30, 2009 Category: Molecular Biology Authors: Yang Y, Hebron HR, Hang J Tags: J Biomol Tech Source Type: journals

Two-Step Versus One-Step RNA-to-C(T) 2-Step and One-Step RNA-to-C(T) 1-Step: Validity, Sensitivity, and Efficiency.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Quantitative RT-PCR can be carried out as a one- or a two-step reaction. However, the choice of method raises controversy from the perspective of the researcher and manufacturer, because of advantages and disadvantages with both systems. We therefore hypothesize that running the RNA-to-C(T) 2-Step kit [(Applied Biosystems (AB), Foster City, CA] using a one-step protocol (as recommended) is not appropriate for quantitation of gene expression levels and should not be performed. Consequently, we ran comparative studies of the two suggested methods to evaluate their efficiency, sensitivity, and accuracy. To ensure precessi...
Source: Journal of Biomolecular Techniques : JBT - June 30, 2009 Category: Molecular Biology Authors: Al-Shanti N, Saini A, Stewart CE Tags: J Biomol Tech Source Type: journals

Association of biomolecular resource facilities survey: service laboratory funding.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In 2007, The Association of Biomolecular Resource Facilities (ABRF) Survey Committee surveyed the ABRF membership and scientists at-large concerning the current state of funding in service-oriented laboratories. Questions pertained to services offered, cost recovery, capital equipment funding, and future outlook. The web-based survey, available for 3 weeks, achieved participation from 209 respondents in 13 countries, 77% of which represented academic laboratories. Most respondents (75%) directed their laboratories. Laboratories depend largely on institutional support and customer recharges to fund operations, but Natio...
Source: Journal of Biomolecular Techniques : JBT - June 30, 2009 Category: Molecular Biology Authors: Ogorzalek Loo R, Nicolet CM, Niece RL, Young M, Simpson JT Tags: J Biomol Tech Source Type: journals

Article watch, july 2009.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, MCG-UGA Medical Partnership, 285 S. Jackson Street, Athens, GA 30602; E-mail: cslaughter@mcg.edu, or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the Association. PMID: 19568458 [PubMed - in process] (Source: Journal of Biomolecular Techniques : JBT)
Source: Journal of Biomolecular Techniques : JBT - June 30, 2009 Category: Molecular Biology Authors: Slaughter C Tags: J Biomol Tech Source Type: journals

Transfer buffer containing methanol can be reused multiple times in protein electrotransfer.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We investigated the feasibility of repeated use of transfer buffer containing methanol in electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to polyvinylidene difluoride (PVDF) membrane using a prestained protein marker of broad molecular sizes. Transfer of the antitumor protein p53 in HEK293T cell extracts, using fresh and used transfer buffer, followed by detection with anti-p53 antibody was also performed to test detectability in immunoblot. Results from these experiments indicate that the transfer buffer can be reused at least five times and maintain a similar extent of protein transfer to ...
Source: Journal of Biomolecular Techniques : JBT - April 1, 2009 Category: Molecular Biology Authors: Pettegrew CJ, Jayini R, Islam MR Tags: J Biomol Tech Source Type: journals

Optimization of a digoxigenin-based immunoassay system for gene detection in Arabidopsis thaliana.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Digoxigenin is derived from a plant steroid hormone digoxin found in the plants Digitalis sp. Digoxigenin has been used successfully in labeling nucleic acids. In this experiment we optimized minimum probe requirement for a nonradioactive digoxigenin-based gene detection system in the model plant Arabidopsis thaliana. We showed that 1 microL of labeled probe was sufficient to hybridize onto 1-10 microg of target plasmid DNA. We also examined the sensitivity of labeled probe and showed that 2 microL of labeled probe was not able to hybridize with 1 microg of target DNA, although 2 microL of labeled probe was able to det...
Source: Journal of Biomolecular Techniques : JBT - April 1, 2009 Category: Molecular Biology Authors: Hart SM, Basu C Tags: J Biomol Tech Source Type: journals

A depletion strategy for improved detection of human proteins from urine.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
With rapidly growing interest in the urine proteome, methods for reducing sample complexity are becoming increasingly important. Depletion strategies for removal of high-abundance proteins from human urine have not been reported. A commercial kit designed for depletion of abundant proteins from plasma was evaluated for removing top proteins from urine of patients with proteinuria. The number of low-abundance proteins identified in urine after depletion increased nearly 2.5-fold. PMID: 19503621 [PubMed - in process] (Source: Journal of Biomolecular Techniques : JBT)
Source: Journal of Biomolecular Techniques : JBT - April 1, 2009 Category: Molecular Biology Authors: Kushnir MM, Mrozinski P, Rockwood AL, Crockett DK Tags: J Biomol Tech Source Type: journals

Quantitative evaluation and selection of reference genes in a rat model of extended liver resection.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Partial hepatectomy (PHx) is a frequently used experimental model for the study of liver regeneration. Real-time quantitative PCR (qPCR) has become the one of the methods of choice for expression profiling of selected genes in order to elucidate the regulation of liver function and regeneration. The expression of five commonly used housekeeping genes (HKGs; Alb, UBC, Hprt, Ywhaz, and GAPDH) were evaluated by qPCR in 70% and 90% rat PHx model at 1, 2, and 7 d after PHx. We set up a closely controlled qPCR procedure validating each critical step and the gene expression stability was statistically evaluated by linear regr...
Source: Journal of Biomolecular Techniques : JBT - April 1, 2009 Category: Molecular Biology Authors: Xing W, Deng M, Zhang J, Huang H, Dirsch O, Dahmen U Tags: J Biomol Tech Source Type: journals

Identification of optimal protocols for sequencing difficult templates: results of the 2008 ABRF DNA Sequencing Research Group difficult template study 2008.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The 2008 ABRF DNA Sequencing Research Group (DSRG) difficult template sequencing study was designed to identify a general set of guidelines that would constitute the best approaches for sequencing difficult templates. This was a continuation of previous DSRG difficult template studies performed in 1996, 1997, and 2003. The distinguishing factors in the present study were the number of DNA templates used, the number of different types of difficult regions tested, and the inclusion of a follow-up phase of the study to identify optimal protocols for each type of difficult template. DNA templates with associated sequencing...
Source: Journal of Biomolecular Techniques : JBT - April 1, 2009 Category: Molecular Biology Authors: Kieleczawa J, Adam D, Bintzler D, Detwiler M, Needleman D, Schweitzer P, Singh S, Steen R, Zianni M Tags: J Biomol Tech Source Type: journals

Polymerase chain reaction preparation of template for massively parallel pyrosequencing.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We report that the use of a biotinylated primer for polymerase chain reaction amplification allows removal of excess primer and poly(A) tract fragments from the sequencing templates, providing much higher yields of useful sequence information from pyrosequencing of amplified templates. This advance allows deep sequencing analysis of nucleic acids isolated from very small tissue samples. Massively parallel pyrosequencing is particularly useful for preliminary investigations of species that have not yet been the subject of significant genomic research, as genomic survey sequences and catalogs of expressed genes provide a mea...
Source: Journal of Biomolecular Techniques : JBT - April 1, 2009 Category: Molecular Biology Authors: Whetten RW, Sofía VA, Frampton J Tags: J Biomol Tech Source Type: journals

Comparison of comparative genomic hybridization technologies across microarray platforms.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This report provides guidance in the selection of platforms based on this wide-ranging evaluation of available CGH platforms. PMID: 19503625 [PubMed - in process] (Source: Journal of Biomolecular Techniques : JBT)
Source: Journal of Biomolecular Techniques : JBT - April 1, 2009 Category: Molecular Biology Authors: Hester SD, Reid L, Nowak N, Jones WD, Parker JS, Knudtson K, Ward W, Tiesman J, Denslow ND Tags: J Biomol Tech Source Type: journals

Toward sustaining women in careers in core laboratories--a workshop review including recommendations from the association of biomolecular resource facilities.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Review of "From Doctorate to Dean or Director: Sustaining Women Through Critical Transition Points in Science, Engineering, and Medicine" (workshop held by the Committee on Women in Science, Engineering, and Medicine of the National Academies, Washington DC, September 18-19, 2008). PMID: 19183800 [PubMed - in process] (Source: Journal of Biomolecular Techniques : JBT)
Source: Journal of Biomolecular Techniques : JBT - December 1, 2008 Category: Molecular Biology Authors: Cilia M Tags: J Biomol Tech Source Type: journals

DNA methylation signature analysis: how easy is it to perform?Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Epigenetic changes, or heritable alterations in gene function that do not affect DNA sequence, are rapidly gaining acceptance as co-conspirators in carcinogenesis. Although DNA methylation signature analysis by methylation-specific polymerase chain reaction has been a breakthrough method in speed and sensitivity for gene methylation studies, several factors still limit its application as a routine diagnostic and prognostic test. PMID: 19183791 [PubMed - in process] (Source: Journal of Biomolecular Techniques : JBT)
Source: Journal of Biomolecular Techniques : JBT - December 1, 2008 Category: Molecular Biology Authors: Piperi C, Farmaki E, Vlastos F, Papavassiliou AG, Martinet N Tags: J Biomol Tech Source Type: journals

Optimization of proteomic sample preparation procedures for comprehensive protein characterization of pathogenic systems.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Mass spectrometry-based proteomics is a powerful analytical tool for investigating pathogens and their interactions within a host. The sensitivity of such analyses provides broad proteome characterization, but the sample-handling procedures must first be optimized to ensure compatibility with the technique and to maximize the dynamic range of detection. The decision-making process for determining optimal growth conditions, preparation methods, sample analysis methods, and data analysis techniques in our laboratory is discussed herein with consideration of the balance in sensitivity, specificity, and biomass losses duri...
Source: Journal of Biomolecular Techniques : JBT - December 1, 2008 Category: Molecular Biology Authors: Mottaz-Brewer HM, Norbeck AD, Adkins JN, Manes NP, Ansong C, Shi L, Rikihisa Y, Kikuchi T, Wong SW, Estep RD, Heffron F, Pasa-Tolic L, Smith RD Tags: J Biomol Tech Source Type: journals

Optimization of Immobilized Gallium (III) Ion Affinity Chromatography for Selective Binding and Recovery of Phosphopeptides from Protein Digests.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Although widely used in proteomics research for the selective enrichment of phosphopeptides from protein digests, immobilized metal-ion affinity chromatography (IMAC) often suffers from low specificity and differential recovery of peptides carrying different numbers of phosphate groups. By systematically evaluating and optimizing different loading, washing, and elution conditions, we have developed an efficient and highly selective procedure for the enrichment of phosphopeptides using a commercially available gallium(III)-IMAC column (PhosphoProfile, Sigma). Phosphopeptide enrichment using the reagents supplied with th...
Source: Journal of Biomolecular Techniques : JBT - December 1, 2008 Category: Molecular Biology Authors: Aryal UK, Olson DJ, Ross AR Tags: J Biomol Tech Source Type: journals

Aptamer selection express: a novel method for rapid single-step selection and sensing of aptamers.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Here we describe a new DNA capture element (DCE) sensing system, based on the quenching and dequenching of a double-stranded aptamer. This system shows very good sensitivity and thermal stability. While quenching, dequenching, and separating the DCE systems made from different aptamers (all selected by SELEX), an alternative method to rapidly select aptamers was developed-the Aptamer Selection Express (ASExp). This process has been used to select aptamers against different types of targets (Bacillus anthracis spores, Bacillus thuringiensis spores, MS-2 bacteriophage, ovalbumin, and botulinum neurotoxin). The DCE system...
Source: Journal of Biomolecular Techniques : JBT - December 1, 2008 Category: Molecular Biology Authors: Fan M, McBurnett SR, Andrews CJ, Allman AM, Bruno JG, Kiel JL Tags: J Biomol Tech Source Type: journals

Systematic internal standard selection for capillary liquid chromatography-mass spectrometry time normalization to facilitate serum proteomics.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Because blood interacts with almost all tissues of the body, it is likely that changes in the overall health of an organism will be reflected in the quantities of specific serum peptides and proteins, making them biomarkers. Due to the complexity of serum, pre-analytical sample simplification and separation are needed prior to mass spectrometric analysis. Use of a reverse-phase capillary column coupled to a mass spectrometer allows for separation and analysis of serum as part of efforts to discover biomarkers. Even after sample simplification by organic solvent precipitation, data files for a single sample typically ex...
Source: Journal of Biomolecular Techniques : JBT - December 1, 2008 Category: Molecular Biology Authors: Merrell K, Thulin CD, Esplin MS, Graves SW Tags: J Biomol Tech Source Type: journals

Optimizing Electroporation Conditions in Primary and Other Difficult-to-Transfect Cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present data demonstrating that by optimizing electroporation parameters, nucleic acid molecules can be delivered in a highly efficient manner. We display transfection results for primary and difficult-to-transfect cell types including human primary fibroblasts, human umbilical vein endothelial cells, Jurkat cells, and two neuroblastoma cell lines [SK-N-SH (human) and Neuro-2A (mouse)] with plasmid DNAs and siRNAs. Our data demonstrate that by determining proper electroporation conditions, glyceraldehyde phosphate dehydrogenase mRNA was silenced in Jurkat cells when compared with negative control siRNA electroporations ...
Source: Journal of Biomolecular Techniques : JBT - December 1, 2008 Category: Molecular Biology Authors: Jordan ET, Collins M, Terefe J, Ugozzoli L, Rubio T Tags: J Biomol Tech Source Type: journals

Effect of Primer Proximity to a Difficult-to-Sequence Region on Read Length and Sequence Quality.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Anecdotal and not well-established evidence implies that there could be some effect of primer proximity in relation to a difficult region on read length and sequence quality. In this paper we sequenced many different categories of difficult regions where primers were located at various distances in relation to such regions and we found that there is only weak, if any, correlation between primer proximity and read length or sequence quality. The occasional improvements observed in some studies could be related instead to more optimal primers or better quality DNA. We suggest that instead of trying to design primers at v...
Source: Journal of Biomolecular Techniques : JBT - December 1, 2008 Category: Molecular Biology Authors: Kieleczawa J Tags: J Biomol Tech Source Type: journals

Systematic method for determining an ideal housekeeping gene for real-time PCR analysis.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
To standardize the amount of biological material between samples (e.g., number of cells or amount of tissue) for quantitative real-time reverse transcriptase PCR (qRT-PCR), the cycle of the target gene at which expression is detected (the cycle threshold, or Ct) is divided by the Ct of a gene either thought to be unaffected by experimental conditions or similarly expressed among donors. Genes that maintain cellular structure or homeostasis, referred to as housekeeping genes, or 18S ribosomal RNA are often used for this purpose. Although unstable or inconsistent housekeeping gene expression will misrepresent experimenta...
Source: Journal of Biomolecular Techniques : JBT - December 1, 2008 Category: Molecular Biology Authors: Mane VP, Heuer MA, Hillyer P, Navarro MB, Rabin RL Tags: J Biomol Tech Source Type: journals

Devitalization of transgenic seed that preserves DNA and protein integrity.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Agricultural biotechnology companies have been asked to provide intact transgenic seed to regulatory agencies as reference materials for evaluating transgene and protein detection methods (PCR and immunoassay). Due to intellectual-property and product-stewardship considerations, submission of devitalized seed prior to regulatory approval is preferable in any given country. Commonly used devitalization procedures, such as heating or autoclaving, degrade the protein and/or DNA rendering the seed unfit as a reference material for these tests. A novel method for devitalizing seed was developed that involves hydration, free...
Source: Journal of Biomolecular Techniques : JBT - December 1, 2008 Category: Molecular Biology Authors: Schafer BW, Cai CQ, Embrey SK, Herman RA, Song P Tags: J Biomol Tech Source Type: journals

Toward sustaining women in careers in core laboratories-a workshop review including recommendations from the association of biomolecular resource facilities.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Review of "From Doctorate to Dean or Director: Sustaining Women Through Critical Transition Points in Science, Engineering, and Medicine" (workshop held by the Committee on Women in Science, Engineering, and Medicine of the National Academies, Washington DC, September 18-19, 2008). PMID: 19183800 [PubMed - in process] (Source: Journal of Biomolecular Techniques : JBT)
Source: Journal of Biomolecular Techniques : JBT - December 1, 2008 Category: Molecular Biology Authors: Cilia M Tags: J Biomol Tech Source Type: journals

Article watch.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Authors: PMID: 19183801 [PubMed - in process] (Source: Journal of Biomolecular Techniques : JBT)
Source: Journal of Biomolecular Techniques : JBT - December 1, 2008 Category: Molecular Biology Tags: J Biomol Tech Source Type: journals

Increased potency and longevity of gene silencing using validated dicer substrates.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Chemically synthesized small interfering RNAs (siRNAs) are tools used for silencing the expression of a single gene. They are mainly employed in basic research applications, but may also have great potential in therapeutic applications. Longer double-stranded RNAs, such as Dicer-substrate 27mers, trigger gene silencing through the intrinsic RNAi pathway. The design of these Dicer-substrate 27mers has been optimized so they can be oriented by Dicer to consistently select the antisense (guide) strand after cleavage to shorter siRNAs, leading to predictable mRNA cleavage. In this paper we describe evidence that these Dice...
Source: Journal of Biomolecular Techniques : JBT - September 1, 2008 Category: Molecular Biology Authors: Hefner E, Clark K, Whitman C, Behlke MA, Rose SD, Peek AS, Rubio T Tags: J Biomol Tech Source Type: journals

Multiplex ligation-dependent probe amplification analysis on capillary electrophoresis instruments for a rapid gene copy number study.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Annotated DNA samples that had been previously analyzed were tested using multiplex ligation-dependent probe amplification (MLPA) assays containing probes targeting BRCA1, BRCA2, and MMR (MLH1/MSH2 genes) and the 9p21 chromosomal region. MLPA polymerase chain reaction products were separated on a capillary electrophoresis platform, and the data were analyzed using GeneMapper v4.0 software (Applied Biosystems, Foster City, CA). After signal normalization, loci regions that had undergone deletions or duplications were identified using the GeneMapper Report Manager and verified using the DyeScale functionality. The result...
Source: Journal of Biomolecular Techniques : JBT - September 1, 2008 Category: Molecular Biology Authors: Jankowski S, Currie-Fraser E, Xu L, Coffa J Tags: J Biomol Tech Source Type: journals

Influence of diet on the proteome of Drosophila melanogaster as assessed by two-dimensional gel electrophoresis and capillary liquid chromatography-mass spectrometry: the hamburger effect revisited.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Proteomic biomarker discovery has been called into question. Diamandis hypothesized that seemingly trivial factors, such as eating a hamburger, may cause sufficient proteomic change as to confound proteomic differences. This has been termed the hamburger effect. Little is known about the variability of complex proteomes in response to the environment. Two methods-two-dimensional gel electrophoresis (2DGE) and capillary liquid chromatography-electrospray ionization time-of-flight mass spectrometry (LCMS)-were used to study the hamburger effect in two cross-sections of the soluble fruit fly proteome. 2DGE measured abunda...
Source: Journal of Biomolecular Techniques : JBT - September 1, 2008 Category: Molecular Biology Authors: Culwell TF, Thulin CD, Merrell KJ, Graves SW Tags: J Biomol Tech Source Type: journals

ABRF-PRG05: De Novo Peptide Sequence Determination.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A common request of proteomics core facilities is protein identification. However, in some instances primary sequence information for the protein in question is not present in public databases. In other cases, the amino acid sequence of a protein may differ in some way from the sequence predicted from the gene sequence in a database as a result of gene mutation, gene splicing, and/or multiple posttranslational modifications. Thus, it may be necessary to determine the sequence of one or more peptides de novo in order to identify and/or adequately characterize the protein of interest. The primary goal of this study was t...
Source: Journal of Biomolecular Techniques : JBT - September 1, 2008 Category: Molecular Biology Authors: Falick AM, Kowalak JA, Lane WS, Phinney BS, Turck CW, Weintraub ST, West KA, Neubert TA Tags: J Biomol Tech Source Type: journals

Molecular Formula and METLIN Personal Metabolite Database Matching Applied to the Identification of Compounds Generated by LC/TOF-MS.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In an effort to simplify and streamline compound identification from metabolomics data generated by liquid chromatography time-of-flight mass spectrometry, we have created software for constructing Personalized Metabolite Databases with content from over 15,000 compounds pulled from the public METLIN database (http://metlin.scripps.edu/). Moreover, we have added extra functionalities to the database that (a) permit the addition of user-defined retention times as an orthogonal searchable parameter to complement accurate mass data; and (b) allow interfacing to separate software, a Molecular Formula Generator (MFG), that ...
Source: Journal of Biomolecular Techniques : JBT - September 1, 2008 Category: Molecular Biology Authors: Sana TR, Roark JC, Li X, Waddell K, Fischer SM Tags: J Biomol Tech Source Type: journals

Miniaturized GPCR Signaling Studies in 1536-Well Format.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
G protein-coupled receptors (GPCRs) are involved in various physiological processes, such as behavior changes, mood alteration, and regulation of immune-system activity. Thus, GPCRs are popular targets in drug screening, and a well-designed assay can speed up the discovery of novel drug candidates. The Promega cAMP-Glo Assay is a homogenous bioluminescent assay to monitor changes in intracellular cyclic adenosine monophosphate (cAMP) concentrations in response to the effect of an agonist, antagonist, or test compound on GPCRs. Together with the Labcyte Echo 555 acoustic liquid handler and the Deerac Fluidics Equator HT...
Source: Journal of Biomolecular Techniques : JBT - September 1, 2008 Category: Molecular Biology Authors: Shultz S, Worzella T, Gallagher A, Shieh J, Goueli S, Hsiao K, Vidugiriene J Tags: J Biomol Tech Source Type: journals

Article watch.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Authors: PMID: 19137118 [PubMed - in process] (Source: Journal of Biomolecular Techniques : JBT)
Source: Journal of Biomolecular Techniques : JBT - September 1, 2008 Category: Molecular Biology Tags: J Biomol Tech Source Type: journals

Highlights of association for biochemistry and molecular biology meeting during experimental biology 2008.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
PMID: 19137100 [PubMed - in process] (Source: Journal of Biomolecular Techniques : JBT)
Source: Journal of Biomolecular Techniques : JBT - July 1, 2008 Category: Molecular Biology Authors: Yadav SP Tags: J Biomol Tech Source Type: journals

Quartz crystal microbalance with dissipation monitoring: enabling real-time characterization of biological materials and their interactions.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This article illustrates some key advances in the development of a special variation of this technique called quartz crystal microbalance with dissipation monitoring (QCM-D). The main feature and advantage of QCM-D, compared with the conventional QCM, is that it in addition to measuring changes in resonant frequency (Deltaf), a simultaneous parameter related to the energy loss or dissipation (DeltaD) of the system is also measured. Deltaf essentially measures changes in the mass attached to the sensor surface, while DeltaD measures properties related to the viscoelastic properties of the adlayer. Thus, QCM-D measures two t...
Source: Journal of Biomolecular Techniques : JBT - July 1, 2008 Category: Molecular Biology Authors: Dixon MC Tags: J Biomol Tech Source Type: journals

Using metabolomics to estimate unintended effects in transgenic crop plants: problems, promises, and opportunities.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Transgenic crops are widespread in some countries and sectors of the agro-economy, but are also highly contentious. Proponents of transgenic crop improvement often cite the "substantial equivalence" of transgenic crops to the their nontransgenic parents and sibling varieties. Opponents of transgenic crop improvement dismiss the substantial equivalence standard as being without statistical basis and emphasize the possible unintended effects to food quality and composition due to genetic transformation. Systems biology approaches should help consumers, regulators, and other stakeholders make better decisions regarding tr...
Source: Journal of Biomolecular Techniques : JBT - July 1, 2008 Category: Molecular Biology Authors: Hoekenga OA Tags: J Biomol Tech Source Type: journals

Population Studies of Intact Vitamin D Binding Protein by Affinity Capture ESI-TOF-MS.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This article discusses the impetus for analyzing intact proteins in a targeted manner across populations and describes the methodology required to couple mass spectrometric immunoassay with electrospray ionization mass spectrometry for the purpose of qualitatively characterizing a prototypical large plasma protein, vitamin D binding protein, across populations. PMID: 19137103 [PubMed - in process] (Source: Journal of Biomolecular Techniques : JBT)
Source: Journal of Biomolecular Techniques : JBT - July 1, 2008 Category: Molecular Biology Authors: Borges CR, Jarvis JW, Oran PE, Rogers SP, Nelson RW Tags: J Biomol Tech Source Type: journals

Selective glutaraldehyde-mediated coupling of proteins to the 3'-adenine terminus of polymerase chain reaction products.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Attachment of proteins to the 3' end of DNA increases stability of the DNA in serum and retards clearance of DNA by major organs, thereby enhancing in vivo half-life and therapeutic potential of DNA. Unfortunately, the length of DNA molecules that can be produced with 3 ' modifications by solid-phase synthesis for protein attachment is limited to 45-60 nucleotides due to uncertainties about sequence fidelity for longer oligonucleotides. Here we describe selective covalent coupling of proteins or other molecules to the 3'-adenine overhang of unlabeled and fluorophore-labeled double-stranded polymerase chain reaction pro...
Source: Journal of Biomolecular Techniques : JBT - July 1, 2008 Category: Molecular Biology Authors: Bruno JG, Crowell R Tags: J Biomol Tech Source Type: journals

Formation of template-switching artifacts by linear amplification.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Linear amplification is a method of synthesizing single-stranded DNA from either a single-stranded DNA or one strand of a double-stranded DNA. In this protocol, molecules of a single primer DNA are extended by multiple rounds of DNA synthesis at high temperature using thermostable DNA polymerases. Although linear amplification generates the intended full-length single-stranded product, it is more efficient over single-stranded templates than double-stranded templates. We analyzed linear amplification over single- or double-stranded mouse H-ras DNA (exon 1-2 region). The single-stranded H-ras template yielded only the i...
Source: Journal of Biomolecular Techniques : JBT - July 1, 2008 Category: Molecular Biology Authors: Chakravarti D, Mailander PC Tags: J Biomol Tech Source Type: journals

Tissue fractionation by hydrostatic pressure cycling technology: the unified sample preparation technique for systems biology studies.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Major bottlenecks in systems biology studies arise from limitations of current sample preparation techniques. Multiple mutually exclusive sample preparation methods, which are often required to extract distinct classes of molecules from cells and tissues, are incompatible with studies of precious or very limited samples. Moreover, the strong detergents and chaotropic agents commonly required to solubilize sample constituents often interfere with subsequent separation and analysis. Here we describe a rapid, detergent-free sample preparation technique that allows efficient concurrent isolation and fractionation of protei...
Source: Journal of Biomolecular Techniques : JBT - July 1, 2008 Category: Molecular Biology Authors: Gross V, Carlson G, Kwan AT, Smejkal G, Freeman E, Ivanov AR, Lazarev A Tags: J Biomol Tech Source Type: journals

Application of atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry for rapid identification of Neisseria species.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI MS) was applied to develop a proteomics-based method to detect and identify Neisseria species. Heat-inactivated clinical isolate cell suspensions of Neisseria gonorrhoeae and strains belonging to five serogroups (A, B, C, W135, and Y) of Neisseria meningitidis were subjected to on-probe protein/peptide extraction and tryptic digestion followed by AP-MALDI tandem MS (MS/MS)-based proteomic analysis. Amino acid sequences derived from three protonated peptides with m/z values of 1743.8, 1894.8, and 1946.8 were identified by AP-MAL...
Source: Journal of Biomolecular Techniques : JBT - July 1, 2008 Category: Molecular Biology Authors: Gudlavalleti SK, Sundaram AK, Razumovski J, Doroshenko V Tags: J Biomol Tech Source Type: journals

High Performance DNA Purification using a Novel Ion Exchange Matrix.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Ion exchange chromatography has emerged as a reliable alternative to classic CsCl-ethidium bromide gradients for isolating nucleic acids of the highest purity. A plasmid purification method based on a unique anion exchange membrane (IEXM) was developed for the production of superior quality plasmids. This method was simpler and more efficient than conventional bead-based methods. Plasmids were extracted from bacterial cells through alkaline lysis. The crude lysate was clarified by a sequential filtration device that not only removed cell debris but micellar aggregates as well. The clarified lysate was mixed with an ext...
Source: Journal of Biomolecular Techniques : JBT - July 1, 2008 Category: Molecular Biology Authors: Yang Y, Hebron HR, Hang J Tags: J Biomol Tech Source Type: journals

Hyphenated tools for phospholipidomics.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The analysis of intact and underivatised lipids in body fluids as well as in cell and tissue extracts is of utmost importance in the field of early diagnosis. Therefore, fast, reliable, and automated analytical methods are needed to detect known as well as unknown species. The combination of solid phase extraction, high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy are best at meeting this challenge. Herein, we show a workflow for the reliable analysis of individual components in phosphatidylethanolamine extracts. The limitations and advantages of the individual metho...
Source: Journal of Biomolecular Techniques : JBT - July 1, 2008 Category: Molecular Biology Authors: Willmann J, Leibfritz D, Thiele H Tags: J Biomol Tech Source Type: journals

The Use of EGFR Exon 19 and 21 Unlabeled DNA Probes to Screen for Activating Mutations in Non-Small Cell Lung Cancer.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Activating mutations in epidermal growth factor receptor-1 (EGFR) are found in 10-15% of Caucasian patients with non-small cell lung carcinoma (NSCLC). Approximately 90% of the mutations are deletions of several amino acids in exon 19 or point mutations in exon 21. Some studies suggest that these mutations identify patients that might benefit from targeted EGFR inhibitor therapy. DNA melting analysis of polymerase chain reaction products can screen for these mutations to identify this patient population. However, amplicon DNA melting analysis, although easily capable of detecting heterozygous mutations by heterodimer f...
Source: Journal of Biomolecular Techniques : JBT - July 1, 2008 Category: Molecular Biology Authors: Willmore-Payne C, Holden JA, Wittwer CT, Layfield LJ Tags: J Biomol Tech Source Type: journals

Article watch.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Authors: PMID: 19137111 [PubMed - in process] (Source: Journal of Biomolecular Techniques : JBT)
Source: Journal of Biomolecular Techniques : JBT - July 1, 2008 Category: Molecular Biology Tags: J Biomol Tech Source Type: journals

Comparing Techniques for the Identification of the MTHFR A1298C Polymorphism.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The restriction fragment length polymorphism (RFLP) technique with the MboII enzyme is used by a number of researchers as a methodology for the identification of the genetic polymorphism MTHFR A1298C. However, the reliability of this enzyme for genotyping this polymorphism has been questioned, since the silent polymorphism T1317C, located close to the polymorphic region A1298C on gene MTHFR, also has a recognition site for MboII. Thus, the fragments formed by the digestion of MboII present similar sizes, making it difficult to differentiate the allele MTHFR 1298A in the presence of the allele MTHFR 1317C. Hence, we inv...
Source: Journal of Biomolecular Techniques : JBT - April 1, 2008 Category: Molecular Biology Authors: de Alvarenga MP, Pavarino-Bertelli EC, Goloni-Bertollo EM Tags: J Biomol Tech Source Type: journals

Immobilized metal affinity electrophoresis: a novel method of capturing phosphoproteins by electrophoresis.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
An immobilized metal affinity electrophoresis (IMAEP) method is described here. In this method, metal ions are immobilized in a native polyacrylamide gel to capture phosphoproteins. The capture of phosphoproteins by IMAEP is demonstrated with immobilized metals like iron, aluminum, manganese, or titanium. In the case studies, phosphoproteins alpha-casein, beta-casein, and phosvitin are successfully extracted from a protein mixture by IMAEP. PMID: 19137092 [PubMed - in process] (Source: Journal of Biomolecular Techniques : JBT)
Source: Journal of Biomolecular Techniques : JBT - April 1, 2008 Category: Molecular Biology Authors: Lee BS, Lasanthi GD, Jayathilaka P, Huang JS, Gupta S Tags: J Biomol Tech Source Type: journals