Screening of PCRP transcription 1 factor antibodies in biochemical assays
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PROVIDER: PRJNA638411 | ENA |
REPOSITORIES: ENA
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Project description:Screening of PCRP transcription factor antibodies in biochemical assays
| PRJNA635402 | ENA
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
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
Project description:The H3D virtual screening cascade contains models for Mycobacterium tuberculosis and Plasmodium falciparum IC50 predictions, as well as ADME, cytotoxicity, and solubility assays.
Model Type: Predictive machine learning model.
Model Relevance: This panel of models provides predictions for the H3D virtual screening cascade.
Model Encoded by: Miquel Duran-Frigola (Ersilia)
Metadata Submitted in BioModels by: Zainab Ashimiyu-Abdusalam
Implementation of this model code by Ersilia is available here:
https://github.com/ersilia-os/eos7kpb
2024-05-13 | MODEL2403270001 | BioModels
Project description: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.
2020-06-09 | GSE151326 | GEO
Project description:Post-transcriptional modifications are important for transfer RNAs (tRNAs) to be efficient and accurate in translation on the ribosome. The m1G37 modification on a subset of tRNAs in bacteria are generated by a conserved methyltransferase TrmD and is essential for bacterial growth. Previous studies showed that m1G37 has an important role in preventing translational frameshifting and also that this modification is coupled with aminoacylation of tRNAs for proline. Here we performed suppressor screening to isolate a mutant E. coli cell that lacks TrmD but is viable, and the whole-genome sequencing revealed several mutations on prolyl-tRNA synthetase (ProRS) gene conferring cell viability in the absence of TrmD. Biochemical assays confirmed uncoupling of m1G37 modification and aminoacylation, and cell-based assays uncovered the critical role of m1G37 in supporting Wobble decoding.
2022-09-24 | E-MTAB-12178 | biostudies-arrayexpress
Project description:This project is aimed at characterizing the interactions of SARS-CoV-2 Spike protein and its variants with multiple full-length antibodies and monitoring the accompanying conformational dynamics. Different categories of antibodies are tested that recognize different domains of the Spike protein. The project aims at identifying the effects of weak, moderate and strong neutralizing antibodies on Spike protein and decipher their mechanisms of action. In addition to the direct binding effects, distal allosteric effects are also determined. A range of biophysical experiments, biochemical assays, and molecular dynamics simulations are used as orthogonal approaches. The rationale is to identify regions on the SARS-CoV-2 Spike protein that acts as indicators for antibody binding and use these hotspots to develop better neutralizing antibodies against SARS-CoV-2 and any future viral pandemics.
2023-10-09 | PXD043818 | Pride
Project description:In recent years, high throughput discovery of human recombinant monoclonal antibodies (mAbs) has been applied, to greatly advance our understanding of the specificity, and functional activity of antibodies, against HIV. Thousands of antibodies have been generated and screened in functional neutralization assays, and antibodies, associated with cross-strain neutralization and passive protection in primates, have been identified. To facilitate this type of discovery, a high throughput-screening tool is needed, to accurately classify mAbs, and their antigen targets. In this study, we analyzed and evaluated a prototype microarray chip, comprised of HIV-1 recombinant proteins gp140, gp120, gp41, and several membrane proximal external region peptides. The protein microarray analysis of 11 HIV-1 envelope-specific mAbs revealed diverse binding affinities and specificities across clades. Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P<0.0001) with concentrations from ELISA binding measurements. Polyclonal immune responses in plasma samples, from HIV-1 infected subjects, exhibited different binding patterns and reactivity against printed proteins. Examining the totality of the specificity of the humoral response in this way reveals the exquisite diversity, and specificity of the humoral response to HIV.
2015-03-10 | GSE66659 | GEO
Project description:Monolignol transport during lignification is a partially solved puzzle: both the mechanism(s) and the transported form of monolignols are unknown in developing xylem of trees. We tested a hypothesis of an active, plasma membrane (PM)-localized transport of monolignol monomers, dimers, and/or glucosidic forms with membrane vesicles prepared of developing xylem, phloem, and lignin-forming, tissue-cultured cells of Norway spruce (Picea abies L. Karst.), as well as of a control material, non-lignifying tobacco (Nicotiana tabacum L.) BY-2 cells. Xylem and BY-2 vesicles transported both coniferin and p-coumaryl alcohol glucoside, but inhibitor assays suggested this transport being over the tonoplast. Based on similar inhibitor assays, lignin-forming, tissue42 cultured cells of spruce had coniferin transport putatively localizing on PM. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis of membrane proteins isolated from spruce developing xylem, phloem and tissue-cultured cells revealed multiple transporters. These were compared to a transporter gene set that was gained by a correlation analysis with a selected set of spruce monolignol biosynthesis genes. Biochemical membrane vesicle assays showed no support for the ABC-transporter-mediated monolignol transport but point to secondary active transporters (such as MFS- or MATE-transporters). In contrast, proteomic and co-expression analyses suggest a role for ABC-transporters and MFS-transporters.
2020-08-14 | PXD017533 | Pride