Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Large-scale evaluation of renewable PRCP monoclonal antibodies by ChIP-exo, STORM, and …

ABSTRACT: The study of chromatin and its organization involves use of antibodies directed against thousands of distinct proteins spanning numerous isoforms and post-translationally modified variants in the human nucleus. Antibodies are essential where epitope-tagging is impractical (e.g., human tissue, cell lines from a wide variety of origins). However, antibody reliability is problematic. The Protein Capture Reagents Program (PCRP) has generated about a thousand renewable monoclonal antibodies that are expected to be more reliable, but these reagents have not been field tested. Since they were generated primarily against chromatin proteins, we tested them in a variety of chromatin-based assays. 887 unique antibodies against 681 unique chromatin targets were assayed by ChIP-exo. A subset were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and PBM protein-DNA interaction assays. While this represents only a small portion of the antibody validation/utility space, and noting that optimization may be needed in each assay, our findings suggest that 10-20% of the PCRP-derived antibodies have utility in a particular biochemical assay. We also describe validation strategies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE151326 | GEO | 2020/06/09

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Screening of PCRP transcription 1 factor antibodies in biochemical assays

Project description:Antibodies offer a powerful means to interrogate specific proteins in a complex milieu, and where epitope tagging is impractical. However, antibody availability and reliability are problematic. The Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies against human-presumptive chromatin proteins in an effort to improve reliability. However, these reagents have not been widely field-tested. We therefore screened their ability in a variety of assays. 887 unique antibodies against 681 unique chromatin proteins, of which 605 are putative sequence-specific transcription factors (TFs), were assayed by ChIP-exo. Subsets were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. At least 6% of the tested antibodies were validated for use in ChIP-based assays by the most stringent of our criteria. An additional 34% produced data suggesting they warranted further testing for clearer validation. We demonstrate and discuss the metrics and limitations to antibody validation in chromatin-based assays.

2020-09-01 | GSE152144 | GEO

Screening of PCRP transcription factor antibodies in biochemical assays

Project description:Antibodies offer a powerful means to interrogate specific proteins in a complex milieu, and where epitope tagging is impractical. However, antibody reliability is problematic. The Protein Capture Reagents Program (PCRP) has generated over a thousand renewable monoclonal antibodies against presumptive chromatin proteins in an effort to improve reliability. However, these reagents have not been widely field tested. We therefore screened them in a variety of chromatin-based assays. 887 unique antibodies against 681 unique chromatin targets were assayed by ChIP-exo. A subset was further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and PBM protein-DNA interaction assays. At least 5% of the tested antibodies were clearly validated by the most stringent of criteria. However, many others produced data that was distinctly different from background, but whose validity was ambiguous. We demonstrate and discuss the metrics and limitation to antibody validation in chromatin-based assays.

2020-06-09 | GSE151287 | GEO

A dataset describing a suite of novel antibody reagents for the RAS signaling network

Project description:RAS genes are frequently mutated in cancer and have for decades eluded effective therapeutic attack. The National Cancer Institute’s RAS Initiative has a focus on understanding pathways and discovering therapies for RAS-driven cancers. Part of these efforts is the generation of novel reagents to enable the quantification of RAS network proteins. Here we present a dataset describing the development, validation (following consensus principles developed by the broader research community), and distribution of 104 monoclonal antibodies (mAbs) enabling detection of 27 phosphopeptides and 69 unmodified peptides from 20 proteins in the RAS network. The dataset characterizes the utility of the antibodies in a variety of applications, including Western blotting, immunoprecipitation, protein array, immunohistochemistry, and targeted mass spectrometry. All antibodies and characterization data are publicly available through the CPTAC Antibody Portal, Panorama Public Repository, and/or PRIDE databases. These reagents will aid researchers in discerning pathways and measuring expression changes in the RAS signaling network.

2019-07-18 | PXD012130 | Pride

Deep LC-MS/MS analysis of the proteome from six cell lines

Project description:Mass spectrometry is a rational orthogonal method for antibody-based assays, but implementation of MS in the validation pipeline of antibody manufacturers is hampered by the high cost and low throughput. Here we present a rapid method for antibody validation based on denaturing gel electrophoresis of biotinylated cell lysates (PAGE) followed by mass spectrometry (MS) and antibody array analysis (MAP). The first step, PAGE, produces 12 fractions containing proteins of increasing molecular weight. The fractions are analyzed in parallel by MS and MAP. Antibodies to be tested are immobilized on color coded polymer beads to create antibody arrays, which can comprise up to several thousand various antibodies. MS data provide definite protein identifications in each fraction, creating a reference for antibody reactivity patterns obtained via MAP. The method employs automated software to compare both datasets and provide validation data for each antibody tested. Due to the high-throughput nature of the assay we were able to screen several thousands of antibodies against six different cell lines. The differences in protein expression between the cell lines provide an additional control of antibody specificity. Using PAGE-MAP it is possible to screen and validate thousands of antibodies in a matter of weeks. Moreover, antibodies are tested under standardized conditions, which allows for direct comparison of their performance.

2018-07-19 | PXD005945 | Pride

A dataset describing a suite of novel antibody reagents for the RAS signaling network

2019-09-03 | PXD012604 | panorama

Genome wide co-localization of Polycomb orthologs and their effects on gene expression in human fibroblasts

Project description:We have identfied antibodies that support chromatin immunoprecipitation (ChIP) of 11 components of Polycomb repressive complex 1 (PRC1) and shown that multiple variants of PRC1 associate with the same DNA. By performing ChIP-seq with antibodies against CBX6, CBX7, CBX8, RING1 and RING2, in two strains of human fibroblasts, we find that all five proteins co-localize at multiple sites in the genome. The binding profiles have inherent architecture that is conserved between the two strains, but there are also a substantial number of differences between the strains, possibly reflecting their anatomical position. The presence of PcG proteins does not equate with gene silencing and their genomic position does not change at senescence. Chromatin immunoprecipitation and sequencing (ChIP-seq) in two strains of human fibroblasts using antibodies against three Pc orthologs (CBX6, CBX7 and CBX8) and both Psc orthologs (RING1 and 2). Validation by ChIP-PCR and re-ChIP. Strand specific RNA-seq and validation by qRT-PCR.

2013-12-19 | E-GEOD-40740 | biostudies-arrayexpress

Escherichia coli strain:887 | isolate:887

Project description:Escherichia coli strain:887 | isolate:887 Genome sequencing

| PRJNA977434 | ENA

Reactivity of commercial antibodies targeting human histone H2B N-terminal tail on the Intel array platform

Project description:The aim of the study was to test the Intel array platform and map the H2B epitopes bound by commercial antibody reagents. We also tested the ability of SET7 PKMT to methylate Intel array peptides. We diluted antibody reagents 1:1000 and incubated dilutions on the silicon-based Intel array platorm.

2011-12-02 | GSE34034 | GEO

Active Promoters give rise to false positive 'Phantom Peaks' in ChIP-Seq experiments

Project description:We applied ChIP-seq to map the chromosomal binding sites for two nucleosome remodeling complexes containing the ATPase ISWI, ACF and RSF, in Drosophila embryos. Employing a panel of polyclonal and monoclonal antibodies directed against their signature subunits, ACF1 and RSF1, robust profiles were obtained indicating that both remodelers co-occupied a large set of active promoters. For further validation we repeated the mapping using chromatin of mutant embryos that do not express ACF1 or RSF1. Surprisingly, the ChIP-seq profiles were unchanged, suggesting that they were not due to specific immunoprecipitation. Conservative analysis lists about 3000 chromosomal loci, mostly active promoters that are prone to non-specific enrichment in ChIP and give rise to ‘Phantom Peaks’. These peaks are not obtained with pre-immune serum and are not prominent in input chromatin. Examination of various ACF1 and RSF1 antibodies in Drosophila melanogaster embryos which are wildtype or mutant for the antibody targets.

2015-07-10 | E-GEOD-67323 | biostudies-arrayexpress

Masison2022 - Cellular ferritin iron storage

Project description:This is a small model of ferritin iron sequestration kinetics. The model’s parameter values were determined from published kinetic and binding experiments and the model was validated against independent data not used in its construction. Beyond its utility to investigate properties of ferritin iron sequestration itself, we propose this model to be used as a building block for larger models of iron metabolism in a variety of specific cell types.

2023-02-07 | MODEL2211030001 | BioModels

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data