Journal of biomolecular techniques : JBT最新文献

{"title":"Development of a Model Protein Interaction Pair as a Benchmarking Tool for the Quantitative Analysis of 2-Site Protein-Protein Interactions.","authors":"A. Yamniuk, J. Newitt, M. Doyle, F. Arisaka, A. M. Giannetti, P. Hensley, D. Myszka, F. Schwarz, J. Thomson, E. Eisenstein","doi":"10.7171/jbt.15-2604-001","DOIUrl":"https://doi.org/10.7171/jbt.15-2604-001","url":null,"abstract":"A significant challenge in the molecular interaction field is to accurately determine the stoichiometry and stepwise binding affinity constants for macromolecules having >1 binding site. The mission of the Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource Facilities (ABRF) is to show how biophysical technologies are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies [such as biosensor, microcalorimetry, or analytic ultracentrifugation (AUC)]. In the present work, the MIRG has developed a robust model protein interaction pair consisting of a bivalent variant of the Bacillus amyloliquefaciens extracellular RNase barnase and a variant of its natural monovalent intracellular inhibitor protein barstar. It is demonstrated that this system can serve as a benchmarking tool for the quantitative analysis of 2-site protein-protein interactions. The protein interaction pair enables determination of precise binding constants for the barstar protein binding to 2 distinct sites on the bivalent barnase binding partner (termed binase), where the 2 binding sites were engineered to possess affinities that differed by 2 orders of magnitude. Multiple MIRG laboratories characterized the interaction using isothermal titration calorimetry (ITC), AUC, and surface plasmon resonance (SPR) methods to evaluate the feasibility of the system as a benchmarking model. Although general agreement was seen for the binding constants measured using solution-based ITC and AUC approaches, weaker affinity was seen for surface-based method SPR, with protein immobilization likely affecting affinity. An analysis of the results from multiple MIRG laboratories suggests that the bivalent barnase-barstar system is a suitable model for benchmarking new approaches for the quantitative characterization of complex biomolecular interactions.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"44 1","pages":"125-41"},"PeriodicalIF":0.0,"publicationDate":"2015-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83021351","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Simultaneous Extraction of Viral and Bacterial Nucleic Acids for Molecular Diagnostic Applications.","authors":"Lauren N Kajiura, Scott D. Stewart, J. Dresios, C. Uyehara","doi":"10.7171/jbt.15-2604-002","DOIUrl":"https://doi.org/10.7171/jbt.15-2604-002","url":null,"abstract":"Molecular detection of microbial pathogens in clinical samples requires the application of efficient sample lysis protocols and subsequent extraction and isolation of their nucleic acids. Here, we describe a simple and time-efficient method for simultaneous extraction of genomic DNA from gram-positive and -negative bacteria, as well as RNA from viral agents present in a sample. This method compared well with existing bacterial- and viral-specialized extraction protocols, worked reliably on clinical samples, and was not pathogen specific. This method may be used to extract DNA and RNA concurrently from viral and bacterial pathogens present in a sample and effectively detect coinfections in routine clinical diagnostics.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"3 1","pages":"118-24"},"PeriodicalIF":0.0,"publicationDate":"2015-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76089075","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines.","authors":"Ryan Muller, M. C. Hammond, D. Rio, Yeon J Lee","doi":"10.7171/jbt.15-2604-003","DOIUrl":"https://doi.org/10.7171/jbt.15-2604-003","url":null,"abstract":"The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"46 1","pages":"142-9"},"PeriodicalIF":0.0,"publicationDate":"2015-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80466667","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Laser-capture microdissection impairs activity-based protein profiles for serine hydrolase in human lung adenocarcinoma.","authors":"S. Collaud, T. Wiedl, E. Cattaneo, A. Soltermann, S. Hillinger, W. Weder, S. Arni","doi":"10.5167/UZH-44238","DOIUrl":"https://doi.org/10.5167/UZH-44238","url":null,"abstract":"Laser-capture microdissection (LCM) enables the selection of a specific and pure cell population from a heterogenous tissue such as tumors. Activity-based protein profiling/profile (ABPP) is a chemical technology using enzyme-specific active site-directed probes to read out the functional state of many enzymes directly in any proteome. The aim of this work was to assess the compatibility of LCM with downstream ABPP for serine hydrolase (SH) in human lung adenocarcinoma. Fresh frozen lung adenocarcinoma tissue was stained with hematoxylin, toluidine blue, or methyl green (MG). Proteome from stained tissue was labeled further with SH-directed probes, and ABPPs were determined on a one-dimensional gel-based approach. This allowed us to assess the impact of staining procedures on their ABPPs. The effect of the LCM process on ABPPs was assessed furthermore using MG-stained lung adenocarcinoma tissue. The staining procedures led to strong changes in ABPPs. However, MG staining seemed the most compatible with downstream ABPP. MG-stained, laser-captured, microdissected tissue showed additional change in profiles as a result of the denaturing property of extraction buffer but not to the microdissection process itself. LCM staining procedures but not microdissection per se interfered with downstream ABPP and led to a strong change in ABPPs of SHs in human lung adenocarcinoma.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"21 1 1","pages":"25-8"},"PeriodicalIF":0.0,"publicationDate":"2010-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80254688","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The ABRF Edman Sequencing Research Group 2008 Study: investigation into homopolymeric amino acid N-terminal sequence tags and their effects on automated Edman degradation.","authors":"R. Thoma, J. Smith, W. Sandoval, J. Leone, P. Hunziker, B. Hampton, K. Linse, N. Denslow","doi":"10.5167/UZH-28072","DOIUrl":"https://doi.org/10.5167/UZH-28072","url":null,"abstract":"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 affinity resin. The addition of the polyhistidine tag presents unique challenges for the determination of protein identity using Edman degradation chemistry. Participating laboratories were asked to sequence one protein engineered in three configurations: with an N-terminal polyhistidine tag; with an N-terminal polyalanine tag; or with no tag. Study participants were asked to return a data file containing the uncorrected amino acid picomole yields for the first 17 cycles. Initial and repetitive yield (R.Y.) information and the amount of lag were evaluated. Information about instrumentation and sample treatment was also collected as part of the study. For this study, the majority of participating laboratories successfully called the amino acid sequence for 17 cycles for all three test proteins. In general, laboratories found it more difficult to call the sequence containing the polyhistidine tag. Lag was observed earlier and more consistently with the polyhistidine-tagged protein than the polyalanine-tagged protein. Histidine yields were significantly less than the alanine yields in the tag portion of each analysis. The polyhistidine and polyalanine protein-R.Y. calculations were found to be equivalent. These calculations showed that the nontagged portion from each protein was equivalent. The terminal histidines from the tagged portion of the protein were demonstrated to be responsible for the high lag during N-terminal sequence analysis.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"3 1","pages":"216-25"},"PeriodicalIF":0.0,"publicationDate":"2009-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84171420","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Hyphenated tools for phospholipidomics.","authors":"J. Willmann, D. Leibfritz, H. Thiele","doi":"10.4172/jpb.s1000223","DOIUrl":"https://doi.org/10.4172/jpb.s1000223","url":null,"abstract":"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 methods are discussed.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"30 1","pages":"211-6"},"PeriodicalIF":0.0,"publicationDate":"2008-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84395365","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Verification of single-peptide protein identifications by the application of complementary database search algorithms.","authors":"James G Rohrbough, Linda A. Breci, Nirav C. Merchant, Susan Miller, P. Haynes","doi":"10.21236/ada439637","DOIUrl":"https://doi.org/10.21236/ada439637","url":null,"abstract":"Data produced from the MudPIT analysis of yeast (S. cerevisiae) and rice (O. sativa) were used to develop a technique to validate single-peptide protein identifications using complementary database search algorithms. This results in a considerable reduction of overall false-positive rates for protein identifications; the overall false discovery rates in yeast are reduced from near 25% to less than 1%, and the false discovery rate of yeast single-peptide protein identifications becomes negligible. This technique can be employed by laboratories utilizing a SEQUEST-based proteomic analysis platform, incorporating the XTandem algorithm as a complementary tool for verification of single-peptide protein identifications. We have achieved this using open-source software, including several data-manipulation software tools developed in our laboratory, which are freely available to download.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"108 1","pages":"327-32"},"PeriodicalIF":0.0,"publicationDate":"2006-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90548458","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Strategies for the synthesis of labeled peptides.","authors":"L. Bibbs, N. Ambulos, S. Kates, A. Khatri, K. Medzihradszky, G. Osapay, S. Weintraub","doi":"10.1007/0-306-46881-6_45","DOIUrl":"https://doi.org/10.1007/0-306-46881-6_45","url":null,"abstract":"","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"5 9","pages":"155-65"},"PeriodicalIF":0.0,"publicationDate":"2000-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91425334","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}