A ChIP-exo screen of 887 Protein Capture Reagents Program transcription factor antibodies in human cells

  1. B. Franklin Pugh1,2
  1. 1Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA;
  2. 2Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA;
  3. 3Division of Genetics, Department of Medicine; Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA;
  4. 4Department of Microbiology and Immunology, Vagelos College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA;
  5. 5Simpson Querrey Institute for Epigenetics and the Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA;
  6. 6EpiCypher Incorporated, Durham, North Carolina 27709, USA;
  7. 7Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA
  • Corresponding authors: fp265{at}cornell.edu, mlbulyk{at}genetics.med.harvard.edu
  • Abstract

    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.

    Footnotes

    • Received March 3, 2021.
    • Accepted July 7, 2021.

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