RT Journal
A1 Lai, William K.M.
A1 Mariani, Luca
A1 Rothschild, Gerson
A1 Smith, Edwin R.
A1 Venters, Bryan J.
A1 Blanda, Thomas R.
A1 Kuntala, Prashant K.
A1 Bocklund, Kylie
A1 Mairose, Joshua
A1 Dweikat, Sarah N.
A1 Mistretta, Katelyn
A1 Rossi, Matthew J.
A1 James, Daniela
A1 Anderson, James T.
A1 Phanor, Sabrina K.
A1 Zhang, Wanwei
A1 Zhao, Zibo
A1 Shah, Avani P.
A1 Novitzky, Katherine
A1 McAnarney, Eileen
A1 Keogh, Michael-C.
A1 Shilatifard, Ali
A1 Basu, Uttiya
A1 Bulyk, Martha L.
A1 Pugh, B. Franklin
T1 A ChIP-exo screen of 887 Protein Capture Reagents Program transcription factor antibodies in human cells
JF Genome Research 
JO Genome Research 
YR 2021 
FD September 01 
VO 31 
IS 9 
SP 1663 
OP 1679 
DO 10.1101/gr.275472.121 
UL http://genome.cshlp.org/content/31/9/1663.abstract 
AB Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability can be problematic, whereas epitope tagging can be impractical in many cases. To address these limitations, the Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies (mAbs) against human presumptive chromatin proteins. However, these reagents have not been widely field-tested. We therefore performed a screen to test their ability to enrich genomic regions via chromatin immunoprecipitation (ChIP) and a variety of orthogonal assays. Eight hundred eighty-seven unique antibodies against 681 unique human transcription factors (TFs) were assayed by ultra-high-resolution ChIP-exo/seq, generating approximately 1200 ChIP-exo data sets, primarily in a single pass in one cell type (K562). Subsets of PCRP mAbs were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. About 5% of the tested antibodies displayed high-confidence target (i.e., cognate antigen) enrichment across at least one assay and are strong candidates for additional validation. An additional 34% produced ChIP-exo data that were distinct from background and thus warrant further testing. The remaining 61% were not substantially different from background, and likely require consideration of a much broader survey of cell types and/or assay optimizations. We show and discuss the metrics and challenges to antibody validation in chromatin-based assays.