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Screening of PCRP transcription factor antibodies in biochemical assays

William K. M. Lai, Luca Mariani, Gerson Rothschild, Edwin R. Smith, Bryan J. Venters, Thomas R. Blanda, Prashant K. Kuntala, Kylie Bocklund, Joshua Mairose, Sarah N Dweikat, Katelyn Mistretta, Matthew J. Rossi, Daniela James, James T. Anderson, Sabrina K. Phanor, Wanwei Zhang, Avani P. Shaw, Katherine Novitzky, Eileen McAnarney, Michael-C. Keogh, Ali Shilatifard, Uttiya Basu, Martha L. Bulyk, B. Franklin Pugh
doi: https://doi.org/10.1101/2020.06.08.140046
William K. M. Lai
1Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA
2Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853
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Luca Mariani
3Division of Genetics, Department of Medicine; Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
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Gerson Rothschild
5Department of Microbiology and Immunology, Vagelos College of Physicians and Surgeons, Columbia University, New York
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Edwin R. Smith
6Simpson Querrey Center for Epigenetics and the Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, 303 East Superior Street, Chicago, IL 60611, USA
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Bryan J. Venters
7EpiCypher Inc., Durham NC 27709
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Thomas R. Blanda
1Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA
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Prashant K. Kuntala
1Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA
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Kylie Bocklund
1Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA
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Joshua Mairose
1Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA
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Sarah N Dweikat
1Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA
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Katelyn Mistretta
1Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA
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Matthew J. Rossi
1Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA
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Daniela James
1Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA
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James T. Anderson
3Division of Genetics, Department of Medicine; Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
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Sabrina K. Phanor
3Division of Genetics, Department of Medicine; Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
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Wanwei Zhang
5Department of Microbiology and Immunology, Vagelos College of Physicians and Surgeons, Columbia University, New York
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Avani P. Shaw
6Simpson Querrey Center for Epigenetics and the Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, 303 East Superior Street, Chicago, IL 60611, USA
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Katherine Novitzky
7EpiCypher Inc., Durham NC 27709
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Eileen McAnarney
7EpiCypher Inc., Durham NC 27709
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Michael-C. Keogh
7EpiCypher Inc., Durham NC 27709
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  • For correspondence: fp265@cornell.edu
Ali Shilatifard
6Simpson Querrey Center for Epigenetics and the Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, 303 East Superior Street, Chicago, IL 60611, USA
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  • For correspondence: fp265@cornell.edu
Uttiya Basu
5Department of Microbiology and Immunology, Vagelos College of Physicians and Surgeons, Columbia University, New York
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  • For correspondence: fp265@cornell.edu
Martha L. Bulyk
3Division of Genetics, Department of Medicine; Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
4Department of Pathology; Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
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  • For correspondence: fp265@cornell.edu
B. Franklin Pugh
1Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA
2Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853
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  • For correspondence: fp265@cornell.edu
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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>

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Screening of PCRP transcription factor antibodies in biochemical assays
William K. M. Lai, Luca Mariani, Gerson Rothschild, Edwin R. Smith, Bryan J. Venters, Thomas R. Blanda, Prashant K. Kuntala, Kylie Bocklund, Joshua Mairose, Sarah N Dweikat, Katelyn Mistretta, Matthew J. Rossi, Daniela James, James T. Anderson, Sabrina K. Phanor, Wanwei Zhang, Avani P. Shaw, Katherine Novitzky, Eileen McAnarney, Michael-C. Keogh, Ali Shilatifard, Uttiya Basu, Martha L. Bulyk, B. Franklin Pugh
bioRxiv 2020.06.08.140046; doi: https://doi.org/10.1101/2020.06.08.140046
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Screening of PCRP transcription factor antibodies in biochemical assays
William K. M. Lai, Luca Mariani, Gerson Rothschild, Edwin R. Smith, Bryan J. Venters, Thomas R. Blanda, Prashant K. Kuntala, Kylie Bocklund, Joshua Mairose, Sarah N Dweikat, Katelyn Mistretta, Matthew J. Rossi, Daniela James, James T. Anderson, Sabrina K. Phanor, Wanwei Zhang, Avani P. Shaw, Katherine Novitzky, Eileen McAnarney, Michael-C. Keogh, Ali Shilatifard, Uttiya Basu, Martha L. Bulyk, B. Franklin Pugh
bioRxiv 2020.06.08.140046; doi: https://doi.org/10.1101/2020.06.08.140046

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