TY - JOUR T1 - Screening of PCRP transcription factor antibodies in biochemical assays JF - bioRxiv DO - 10.1101/2020.06.08.140046 SP - 2020.06.08.140046 AU - William K. M. Lai AU - Luca Mariani AU - Gerson Rothschild AU - Edwin R. Smith AU - Bryan J. Venters AU - Thomas R. Blanda AU - Prashant K. Kuntala AU - Kylie Bocklund AU - Joshua Mairose AU - Sarah N Dweikat AU - Katelyn Mistretta AU - Matthew J. Rossi AU - Daniela James AU - James T. Anderson AU - Sabrina K. Phanor AU - Wanwei Zhang AU - Avani P. Shaw AU - Katherine Novitzky AU - Eileen McAnarney AU - Michael-C. Keogh AU - Ali Shilatifard AU - Uttiya Basu AU - Martha L. Bulyk AU - B. Franklin Pugh Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/06/09/2020.06.08.140046.abstract N2 - 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.Competing Interest StatementM.L.B. is a co-inventor on U.S. patent #8,530,638 on universal PBM technology. BFP has a financial interest in Peconic, LLC, which utilizes the ChIP-exo technology implemented in this study and could potentially benefit from the outcomes of this research. EpiCypher is a commercial developer of reagents to support CUTANA CUT&RUN. The authors in the Basu and Shilatifard labs declare no competing financial interests. ER -