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Joint Ressearch Group Presentation on Proteomics (PRG, sPRG and iPRG)

Turck, C.; Friedman, D.; Rudnick, P.; Farmar, J.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /09/2010 Português
Relevância na Pesquisa
57.59368%
r4-1

Genomic Variation Research Group (GVRG): Analysis of Copy Number Variation in C. elegans using Next Generation Sequencing

Kingham, B.; Blake, S.; Noll, A.; Dagnall, C.; Escobar, H.; Hutchinson, A.; Forrester, J.; Lytle, C.; Jonscher, K.; Sanderson, B.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /09/2010 Português
Relevância na Pesquisa
67.690215%
r5b

Nucleic Acid Research Group (NARG) 2009-2010 Study : Optimal Priming Strategies for cDNA Synthesis in Real-Time RT-qPCR

Hunter, T.C.; Knudtson, K.L.; Nadella, V.; Sol-Church, K.; Taylor, W.L.; Tighe, S.; Yueng, A.T.; Chittur, S.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /09/2010 Português
Relevância na Pesquisa
67.690215%
r1-1

iPRG 2011: A Study on the Identification of Electron Transfer Dissociation (ETD) Mass Spectra

Askenazi, M.; Bandeira, N.; Chalkley, R.J.; Clauser, K.R.; Deutsch, E.; Lam, H.H.N.; McDonald, W.H.; Neubert, T.; Rudnick, P.A.; Martens, L.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /10/2011 Português
Relevância na Pesquisa
67.740776%
The field of mass spectrometry based proteomics has seen several key innovations over the last several years, including novel experimental methods, new instruments, and unique fragmentation strategies. The latter, in the form of electron capture dissociation (ECD) and electron transfer dissociation (ETD) have captured the imaginations of many researchers, expanding their ability to identify and analyze peptides and proteins. However, since ECD/ETD spectra differ substantial from more traditional collision induced dissociation (CID) spectra in both their prominent ion series as well as their preferred bond-breaking characteristics, the (automatic) interpretation of ECD/ETD spectra requires novel algorithm optimizations. Efficient identification of ECD/ETD spectra thus remains an active and exciting field of proteomics informatics research. In this work, the ABRF Proteome Informatics Research Group (iPRG) presents the results of a collaborative study focusing on the analysis of an LC-MS/MS dataset from a yeast lysate digested with Lys-C and enriched for highly charged peptides using strong cation exchange fractionation. The data derived from one fraction analyzed exclusively by ETD was distributed to participants for analysis in several equivalent formats...

PRG-2011: Defining the Interaction between Users and Suppliers of Proteomics Services

Andacht, T.M.; Bunger, M.K.; Bystrom, C.; Dangott, L.; Molina, H.; Moritz, R.L.; Settlage, R.E.; Turck, C.W.; Hawke, D.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /10/2011 Português
Relevância na Pesquisa
67.690215%
Over the last ten years the Proteomics Research Group (PRG) has undertaken technical studies that have covered a wide range of issues unique to the rapidly developing field of proteomics and have included a range of qualitative and quantitative experiments. The PRG studies have resulted in a great deal of attention not only within the ABRF community but also outside as is evident from numerous articles dealing with proteomics methods, procedures and standardization. As the field continues to develop, the diversity of instrumentation and laboratory workflows have grown in tandem. Therefore, in the PRG2011 study it seemed especially useful to perform a survey to help the PRG define future studies based on the current blend of sample types and technologies and obtain a view of emerging trends. A survey was created to ascertain three main insights into core facility function: 1) How labs interact with their clients, 2) The capacity of labs to meet the demands of their clients, and 3) The blend of experimental techniques offered to and requested by clients. Survey questions were designed to obtain information from both users of core facilities and the directors and personnel of core facilities. Questions covered such topics as the type and age of instruments in use...

PERG Research Group Presentation: Refolding Study

Kinsland, C.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /10/2011 Português
Relevância na Pesquisa
87.817%
Currently, the overwhelming majority of protein purification projects start with a recombinant protein expressed in a suitable host. For a range of reasons, E. coli is the predominant expression host yet a large percentage of proteins expressed therein are found in an insoluble form called inclusion bodies. Inclusion bodies have the advantage of consisting of relatively homogeneous protein, which can simplify the purification process. However, this leaves the challenge of solubilizing and refolding the protein into its native and biologically active structure. The conditions for efficient refolding are particular for each protein and there are a wide range of methods to choose from. For this and other reasons, many researchers are hesitant to pursue a refolding strategy to obtain a target protein. An overview of refolding methods and strategies will be presented along with a description of the upcoming Protein Expression Research Group (PERG) refolding study.

gPRG: Toward Consensus on Glycan Analysis: Reliable Methods and Reproducibility

Kolarich, D.; Orlando, R.; Zaia, J.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /10/2011 Português
Relevância na Pesquisa
67.690215%
The field has undertaken over the past few years a series of studies to evaluate methods for glycan analysis. There are several methods that are widely used at this time for characterization of glycoprotein glycans that appear to be generally reliable. These include (1) reductive amination followed by reversed phase chromatography, (2) reductive amination with matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS), (3) reductive amination with electrospray liquid chromatography-MS, and (4) permethylation with tandem MS. The ABRF glycoprotein research group (gPRG) is planning a study in which participant laboratories carry out glycan analysis according to protocols representing best practice in the field. The results will be presented at the 2012 ABRF meeting. The results will be useful for demonstrating the reproducibility of glycan analysis using comparable protocols. They will also highlight the extent to which different methods bias the glycan analysis results obtained. This workshop will summarize the history and use of the glycan analysis methods proposed for the 2012 study. There will be opportunity for discussion and comment on these methods.

Microarray Research Group Projects, 2010–11

Reyero-Vinas, N.G.; Jafari, N.; Baldwin, D.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /10/2011 Português
Relevância na Pesquisa
77.740776%
Members of the MARG will discuss our research projects: Comparison of microarray and deep sequencing platforms for microRNA profiling, Performance of a synthetic human microRNA reference panel, Participation in the SEQC Sequencing Quality Control consortium, and RNA-Seq profiling of environmental samples exposed to the Gulf oil spill.

Metabolomics Research Group 2011 Study

Asara, J.M.; Tolstikov, V.V.; Aronov, P.; Kesler, B.; Shulaev, V.; Turck, C.W.; Wikoff, W.R.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /10/2011 Português
Relevância na Pesquisa
87.93133%
The ABRF Metabolomics Research Group (MRG) was formed in 2009 and aims to educate research scientists and resource facilities in the analytical approaches and management of data resulting from comprehensive metabolite studies and to promote the science and standardization of metabolomic analyses for a variety of applications. Last year the MRG conducted a ‘Survey Study’ on the current use of metabolomics technologies and procedures in core facilities. This year the MRG is organizing a ‘Research Study’ involving a spiked plasma sample. The study sample consists of a human biofluid as the matrix, replicating a typical small scale metabolomics pilot experiment that either a core or research laboratory would perform. The sample consists of two groups of normal human plasma (NIST plasma ‘Standard Reference Material’) with spiked in compounds. There are three biological replicates in each group (n = 3 design) with different levels of spiked compounds differentiating the two groups. Participants are asked to determine statistical significance, fold change, and identify compounds that differ significantly between groups A and B. The design reflects issues encountered in an actual metabolomics experiment conducted with human or animal specimens. The study is compatible with many methodological approaches in metabolomics...

iPRG-2013: Proteome Informatics Research Group Study: Using RNA-Seq Data to Refine Proteomic Data Analysis

Bandeira, N.; Chambers, M. C.; Cottrell, J. S.; Deutsch, E. W.; Kapp, E. A.; Lam, H. H. N.; Neubert, T. A.; Sun, R-X.; Vitek, O.; Weintraub, S. T.; Chalkley, Robert
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /05/2013 Português
Relevância na Pesquisa
67.690215%
The Proteome Informatics Research Group (iPRG) this year performed a study to evaluate the benefits of using databases derived from RNA-Seq data for peptide identification. The proteomic dataset provided consisted of high mass accuracy tandem mass spectra acquired when analyzing human peripheral blood mononuclear cells. A variety of different types of sequence databases were supplied. These included a standard protein sequence database; a database containing only sequences of proteins expressed in the sample based on RNA-Seq data; a database that included sequence and splice variants; a database of sequences that could not be reconciled to known expressed gene sequences.

The New ABRF Flow Cytometry Research Group (FCRG)

Box, Andrew; Chittur, Sridar; Cochran, Matthew; DeLay, Monica; Lopez, Peter; Meyer, Michael; Neubert, Thomas; Pletcher, Hank; Tighe, Scott; Bergeron, Alan
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /05/2013 Português
Relevância na Pesquisa
67.771895%
The Flow Cytometry Research Group (FCRG) is the latest addition to the ABRF RG family. This RG is currently in its first year and has 9 members several of whom are new to the ABRF but have been very active in and come from the flow cytometry core community. The FCRG has submitted a 3 year research plan that will characterize alterations in both gene expression and ultimately cellular function as a result of the stresses imparted by cell sorting. We will use a variety of cell types, lasers, and sorters to identify optimal conditions and eventually Best Practices for minimal cellular system disruptions. Integration of flow cytometry with other core technologies and ABRF RGs will become even more critical as many new technologies will fully take advantage of the sample processing capability of cell sorting allowing higher resolution targeted downstream molecular applications such as single cell gene expression. The new FCRG will seek to foster collaboration, integration and synergy between experts of diverse technologies the very factors that will become increasingly vital to successful research.

Genomics Research Group (GRG): Elucidating the Effects of the Deepwater Horizon Oil Spill on the Atlantic Oyster Using Global Transcriptome Analysis

Raghavachari, Nalini; Showmaker, Kurt; Liu, Poching; Jafari, Nadereh; Barker, Natalie; Willett, Kristine L.; Corrales, Jone; Patterson, Heather K.; Carmichael, Ruth H.; Baldwin, Don; Reyero, Natalia G.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /05/2013 Português
Relevância na Pesquisa
67.690215%
Global transcriptome analysis is of growing importance in understanding how altered expression of genetic variants contributes to complex diseases such as cancer, diabetes, and heart disease as well as the effect of environmental pollutants to living organisms. The Genomics Research Group applied next generation sequencing technologies to study the effects of Deep Water Horizon oil spill on the transcriptome of atlantic oysters. The Deep Water Horizon oil spill resulted in the release of over 200 million gallons of crude oil into the waters of the Gulf of Mexico. Over two million gallons of chemical were used to emulsify and disperse oil plumes posing further risks to the environment in addition to the direct impacts of crude oil. Biota such as the commercially important Atlantic oyster Crassostrea virginica, were inevitably exposed to spill-related contaminants in the Gulf. The potential effects of oiled water and sediments on oysters range from non-detectable to reduced settlement to impaired immune function, acute intoxication, and death due to bioaccumulation of contaminants. Oil also may affect oxygen diffusion through the water column, and in some cases lead to hypoxic conditions that prompt avoidance migration by mobile species. Sedentary organisms such as oysters are even more susceptible to these negative effects of oil contamination. The mechanisms of toxicity of the oil and spill-related compounds are not well understood. In order to understand these mechanisms...

Glycoprotein Research Group (gPRG) 2013 Interlaboratory Study: Quantitative N-glycan Profiling of Prostate Specific Antigen

Thaysen-Andersen, Morten; Suckau, Detlev; Karen, Jonscher; Kolarich, Daniel; Orlando, Ron; Mccomb, Mark; Zaia, Joseph; Leymarie, Nancy
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /05/2013 Português
Relevância na Pesquisa
57.54469%
The fact that most proteins are glycosylated underlies the key roles played by glycosylation during evolution. The functions of glycoproteins are tremendously broad and include cell attachment recognition, homeostasis, transport of molecules, and enzymatic and immunology recognition domains. One of the principal axes of glycoprotein research is to understand better the correlation between glycan structure and function. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information.

Genomics Research Group Session

Baldwin, D.; Chittur, S. V.; Raghavachari, N.; Jafari, N.; Aquino, C.; Perera, A.; Reyero, N.G.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /05/2014 Português
Relevância na Pesquisa
68.20126%
The Genomics Research Group (GRG) presentation is intended to describe the current activities of the group in applying the latest tools and technologies for transcriptome analysis to determine the advantages and disadvantages of each of the platforms. We will present three ongoing projects. In the first project, we specifically evaluated microarrays, QPCR and Next Generation Sequencing (NGS) platforms for examining the sensitivity and specificity of microRNA detection using synthetic miRNA standards. In a second study, we used total RNA from Atlantic oyster samples to analyze the transcriptome and study the effect of oil spill and hypoxia as secondary stressor using next generation sequencing. Furthermore, we are now in the process of assembling the Atlantic oyster genome in collaboration with the Genomics Bioinformatics Research Group (GBIRG). We will also present our new project, which will involve the comparison of gene expression profiles of individual SUM149PT cells treated with the histone deacetylase inhibitor TSA vs. untreated controls. The goals of this project are to demonstrate RNA sequencing (RNA-seq) methods for profiling the ultra-low amounts of RNA present in individual cells, and to demonstrate the use of the Fluidigm system for cell capture and RNA amplification in C1 Single-Cell Auto Prep Integrated Fluidic Circuits (IFCs). In this session...

Metabolomics Research Group (MRG)

Turck, C.; Dodder, N.; Kesler, B.; Wikoff, W. R.; O'Connell, T.M.; Aronov, P.; Tolstikov, V.V.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /09/2010 Português
Relevância na Pesquisa
67.690215%
r3-0

Conclusions from the MIRG 2010 Benchmark Study: Molecular Interactions in a Three Component System and Presentation of 2011 Survey Results on Label-Free Technologies

Yadav, S.P.; Bergqvist, S.; Doyle, M.L.; Eisenstein, E.; Robinson, M.K.; Neubert, T.; Yamniuk, A.P.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /10/2011 Português
Relevância na Pesquisa
67.740776%
Characterizing the assembly of multi-protein complexes and the competition between multiple protein ligands for a given target are common challenges faced by core facilities. The MIRG2010 Benchmark study was designed to assess participants' ability to correctly describe the interactions between two protein ligands and their target protein using primarily biosensor technologies such as surface Plasmon resonance. Participants were provided with microgram quantities of three proteins (A, B and C) and asked to determine if a ternary A-B-C complex can form, or if ligands B and C bind competitively to protein A. This presentation will summarize the conclusions from the 2010 Benchmark Study, and provide perspective on the potential for future application of this system as a reference standard for quantitative characterization of protein-protein interactions using biosensor technologies. The field of label-free biophysical technologies like surface plasmon resonance (SPR) and isothermal calorimetry (ITC) are becoming indispensable in translational research and in the discovery phase of biotherapeutics. Investigators are much more aware about the developments in biomolecular interaction analysis using SPR and ITC and usefulness of these technologies in designing better drugs based on biomolecules and vaccines. The Molecular Interaction Research Group (MIRG) of ABRF has conducted an on-line survey to capture the recent explosive developments in these technologies. The survey was targeted to both academia and pharmaceutical industry and the survey data will be presented during the meeting.

Antibody Technology Research Group (ARG) Presentation of Studies and Initiatives

Agnew, Brian; Weis-Garcia, Frances; Carnahan, Robert
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /05/2013 Português
Relevância na Pesquisa
68.307026%
The Antibody-technologies Research Group (ARG) was formed to foster a community of individuals focused on developing and using antibodies as well as to educate research scientists about the generation, production, manipulation, and optimization of research grade antibodies. In line with these aims, ARG undertook the following activities, which will be discussed at this session. Firstly, a study was conducted to compare of the impact of various antibody-labeling approaches on the ultimate antibody performance. Three distinct antibody-labeling approaches were compared to determine their impact on the functionality of antibodies previously determined to be “difficult to label”. Results suggest a novel click-chemistry based approach may be superior for difficult labeling situations. Secondly, a multi-year study comparing the effectiveness of various immunization strategies was begun. Several approaches to developing a robust humoral immune response were compared head-to-head across multiple sites. Preliminary results have identified adjuvant formulations that drive a stronger antibody respnse than others. Finally, we will review the progress and challenges associated with the electronic repository for antibody related protocols and unpublished lessons learned from working with antibodies. This session will conclude with a discussion on the research group's efforts for the coming year.

Results of the 2010 Glycoprotein Research Group's (gPRG) Study on Quantitative Glycoprotein Analysis

Orlando, R.; Leymarie, N.; Keck, R.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /09/2010 Português
Relevância na Pesquisa
57.54469%
r6-1

ABRF-sPRG2011 Study: Development of a Comprehensive Standard for Analysis of Post-Translational Modifications

Colangelo, C.; Dufresne, C.P.; Farmar, J.G.; Friedman, D.B.; Kinsinger, C.; Lilley, K.S.; Mechtler, K.; Phinney, B.S.; Shaffer, S.A.; Weintraub, S.T.; Ivanov, A.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /10/2011 Português
Relevância na Pesquisa
67.690215%
The Proteomics Standards Research Group (sPRG) has initiated a study that focuses on development of a standard that can be used in both assessment of a laboratory's ability to detect an array of post-translational modifications in a complex proteomic sample and development of new approaches for characterization of post-translationally modified proteins. The sample that has been generated for the first stage of this study contains a mixture of more than seventy synthetic peptides with a variety of modifications, in a tryptic digest of six proteins. Modifications represented in the sample include acetylation, methylation, nitration, phosphorylation, and sulfation. The individual proteins were purified, digested and analyzed prior to formulation of the sample. The synthetic peptides were each analyzed and then mixed with the digests; the mixture was aliquoted and lyophilized in sufficient quantities that would permit evaluation by several analytical approaches. The sample was fully characterized by members of the sPRG. It is planned that in 2011, the sample will be distributed to requesting laboratories for a larger study focusing on PTM characterization. The sPRG will present results obtained at different stages of preparation and characterization of the sample. Approaches for analysis of complex proteomic samples containing various post-translationally modified proteins will also be discussed. The sPRG is planning to open the study for sample requests at the ABRF 2011 conference. From the responses returned by the participating laboratories...

Determining miRNA Expression Levels in Degraded RNA Samples Using Real-Time RT-qPCR and Microarray Technologies

Tighe, S.; Holbrook, J.; Nadella, V.; Carmical, R.; Sol-Church, K.; Yueng, A.T.; Chittur, S.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /10/2011 Português
Relevância na Pesquisa
67.817%
The Nucleic Acid Research Group (NARG) has previously conducted studies evaluating the impact of RNA integrity and priming strategies on cDNA synthesis and real-time RT-qPCR. The results of last year's field study as it relates to degraded RNA will be presented. In continuation of the RNA integrity theme, this year's study was designed to evaluate the impact of RNA integrity on the analysis of miRNA expression using real-time RT-qPCR. Target section was based on data obtained by the Microarray Research Group (MARG) and other published data from next gen sequencing. These 9 miRNAs represent three groups of miRNA that are expressed at low, medium or high levels in the First Choice human brain reference RNA sample. Two popular RT priming strategies tested in this study include the Megaplex miRNA TaqMan assay (ABI) and the RT2 miRNA qPCR assay (Qiagen/SA Biosciences). The basis for the ABI assay design is a target-specific stem-loop structure and reverse-transcription primer, while the Qiagen design combines poly(A) tailing and a universal reverse transcription in one cDNA synthesis reaction. For this study, the human brain reference RNA was subject to controlled degradation using RNase A to RIN (RNA Integrity Number) values of 7 (good)...