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Recombinant antibodies to histone post-translational modifications

Nature Methods volume 10, pages 992–995 (2013)Cite this article

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Abstract

Variability in the quality of antibodies to histone post-translational modifications (PTMs) is a widely recognized hindrance in epigenetics research. Here, we produced recombinant antibodies to the trimethylated lysine residues of histone H3 with high specificity and affinity and no lot-to-lot variation. These recombinant antibodies performed well in common epigenetics applications, and enabled us to identify positive and negative correlations among histone PTMs.

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Figure 1: Characterization of commercial and recombinant anti-H3K9me3 antibodies.
Figure 2: Validation and utility of high specificity of recombinant antibodies.

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Acknowledgements

We thank K.D. Wittrup (Massachusetts Institute of Technology) for an antibody library, J. Lavinder and G. Georgiou for assistance in peptide procurement, J. Ahringer (Cambridge University) for sharing commercial antibodies, M. Gardel and Y. Beckham (University of Chicago) for the NIH 3T3 cell line and advice, A.A. Kossiakoff and M. Lugowski for access to cell culture equipment and microscopes, R. Hoey, N. Bharwani, A. Crofts and R. Ptashkin for technical assistance, S. Nishikori for helpful discussion, and members of the University of Chicago DNA sequencing and flow cytometry core facilities, and of the Institute for Genomics and Systems Biology High-throughput Genome Analysis facility. This work was supported by US National Institutes of Health grants (R21 DA025725 and RC1 DA028779 to S.K., GM085394 to B.D.S., U01 HG004264 to K.P.W. and U01 ES017154 to P.J.F.), and the University of Chicago Comprehensive Cancer Center (to S.K.).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois, USA

    Takamitsu Hattori, Joseph M Taft, Kalina M Swist, Akiko Koide, Alexander J Ruthenburg & Shohei Koide

  2. Department of Biochemistry, Wrocław University of Technology, Wrocław, Poland

    Kalina M Swist

  3. Ben May Department of Cancer Research, The University of Chicago, Chicago, Illinois, USA

    Hao Luo & Yingming Zhao

  4. Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, California, USA

    Heather Witt & Peggy J Farnham

  5. Institute for Genomics and Systems Biology, The University of Chicago, Chicago, Illinois, USA

    Matthew Slattery & Kevin P White

  6. Department of Human Genetics, The University of Chicago, Chicago, Illinois, USA

    Matthew Slattery & Kevin P White

  7. Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois, USA

    Alexander J Ruthenburg

  8. Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA

    Krzysztof Krajewski & Brian D Strahl

Authors
  1. Takamitsu Hattori
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  14. Shohei Koide
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Contributions

T.H. characterized commercial antibodies; T.H., A.K. and S.K. designed phage-display libraries and selection schemes; T.H., J.M.T. and K.M.S. performed selections and biophysical characterization of recombinant antibodies; K.K. and B.D.S. designed and made synthetic peptides; T.H., H.W., M.S., A.J.R., K.P.W., P.J.F. and S.K. designed ChIP experiments; T.H., H.W. and M.S. conducted ChIP experiments and data analysis; T.H., H.L., Y.Z. and S.K. designed IP-MS experiments; T.H. and H.L. performed IP-MS and data analysis: T.H. and S.K. designed HMT assay; T.H. conducted HMT assay. T.H. and S.K. wrote the manuscript. All authors commented on the manuscripts.

Corresponding author

Correspondence to Shohei Koide.

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Competing interests

T.H., J.M.T., A.K. and S.K. are named as inventors in a provisional patent application (61/839,972) filed by the University of Chicago on the described materials.

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Supplementary Figures 1–6 and Supplementary Note (PDF 3321 kb)

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Hattori, T., Taft, J., Swist, K. et al. Recombinant antibodies to histone post-translational modifications. Nat Methods 10, 992–995 (2013). https://doi.org/10.1038/nmeth.2605

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