BioProject

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. More...

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. Overall design: We performed CUT&RUN usking K562 extracted nuclei on 40 TFs using 40 PCRP sourced antibodies Less...