BioProject

Antibodies offer a powerful means to interrogate specific proteins in a complex milieu, and where epitope tagging is impractical. More...

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. Overall design: ChIP-sequencing in HCT116 cells for hybridomas generated by NIH's Protein Capture Reagent Program. For NRF1, two hybridomas were tested with and without RNAi. For HSF1, seven hybridomas were tested at both 37 degrees and after 1 hour of heat shock (43°C). Less...