Abstract
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 Statement
M.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.
Footnotes
Contact Information: William KM Lai <wkl29{at}cornell.edu>, Luca Mariani <lucamariani77{at}gmail.com>, Gerson Rothschild <ger2{at}cumc.columbia.edu>, Edwin R. Smith <edwinsmith{at}northwestern.edu>, Bryan J. Venters <bventers{at}epicypher.com>, Thomas R Blanda <trb5344{at}psu.edu>, Prashant K Kuntala <pxk284{at}psu.edu>, Kylie Bocklund <kxb357{at}psu.edu>, Joshua Mairose <josh.mairose{at}gmail.com>, Sarah N Dweikat <szd41{at}psu.edu>, Katelyn Mistretta <katelynsmistretta{at}gmail.com>, Matthew J. Rossi <mjr26{at}psu.edu>, Daniela James <daniela.james{at}psu.edu>, James T. Anderson <jamesanderson12358{at}gmail.com>, Sabrina K. Phanor <sphanor{at}bwh.harvard.edu>, Wanwei Zhang <wz2370{at}cumc.columbia.edu>, Avani P. Shaw <avani.shah1{at}northwestern.edu>, Katherine Novitzky <knovitzky{at}epicypher.com>, Eileen McAnarney <emcanarney{at}epicypher.com>, Michael-C. Keogh <mkeogh{at}epicypher.com>, Ali Shilatifard <ash{at}northwestern.edu>, Uttiya Basu <ub2121{at}cumc.columbia.edu>, Martha L. Bulyk <mlbulyk{at}genetics.med.harvard.edu>, B. Franklin Pugh <fp265{at}cornell.edu>
