Skip to main content
SpringerLink
Log in
Book cover

Histones pp 149–164Cite as

Production and Purification of Antibodies Against Histone Modifications

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1528))

Abstract

Antibodies that recognize specific histone modifications are invaluable tools to study chromatin structure and function. There are numerous commercially available antibodies that recognize a remarkable diversity of histone modifications. Unfortunately, many of them fail to work in certain applications or lack the high degree of specificity required of these reagents. The production of affinity-purified polyclonal antibodies against histone modifications demands a little effort but, in return, provides extremely valuable tools that overcome many of the concerns and limitations of commercial antibodies. We present a series of protocols and guidelines for the production and use of large amounts of polyclonal antibodies that recognize modifications of canonical histones. Our protocols can be applied to obtain antibodies that occur in histone variants and proteins other than histones. In addition, some of our protocols are compatible with the production of monoclonal or recombinant antibodies.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   159.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Brumbaugh J, Phanstiel D, Coon JJ (2008) Unraveling the histone’s potential: a proteomics perspective. Epigenetics 3:254–257

    Article  PubMed  PubMed Central  Google Scholar 

  2. Egelhofer TA, Minoda A, Klugman S, Lee K, Kolasinska-Zwierz P, Alekseyenko AA et al (2010) An assessment of histone-modification antibody quality. Nat Struct Mol Biol 18:91–93

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Fuchs SM, Strahl BD (2011) Antibody recognition of histone post-translational modifications: emerging issues and future prospects. Epigenomics 3:247–249

    Article  CAS  PubMed  Google Scholar 

  4. Nishikori S, Hattori T, Fuchs SM, Yasui N, Wojcik J, Koide A et al (2012) Broad ranges of affinity and specificity of anti-histone antibodies revealed by a quantitative peptide immunoprecipitation assay. J Mol Biol 424:391–399

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Rothbart SB, Lin S, Britton LM, Krajewski K, Keogh MC, Garcia BA et al (2012) Poly-acetylated chromatin signatures are preferred epitopes for site-specific histone H4 acetyl antibodies. Sci Rep 2:489

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Tjeertes JV, Miller KM, Jackson SP (2009) Screen for DNA-damage-responsive histone modifications identifies H3K9Ac and H3K56Ac in human cells. EMBO J 28:1878–1889

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Rothbart SB, Dickson BM, Raab JR, Grzybowski AT, Krajewski K, Guo AH et al (2015) An interactive database for the assessment of histone antibody specificity. Mol Cell 59:502–511

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Guillemette B, Drogaris P, Lin HH, Armstrong H, Hiragami-Hamada K, Imhof A et al (2011) H3 lysine 4 is acetylated at active gene promoters and is regulated by H3 lysine 4 methylation. PLoS Genet 7, e1001354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Drogaris P, Villeneuve V, Pomies C, Lee EH, Bourdeau V, Bonneil E et al (2012) Histone deacetylase inhibitors globally enhance h3/h4 tail acetylation without affecting h3 lysine 56 acetylation. Sci Rep 2:220

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Baker M (2015) Reproducibility crisis: blame it on the antibodies. Nature 521:274–276

    Article  CAS  PubMed  Google Scholar 

  11. Hirota T, Lipp JJ, Toh BH, Peters JM (2005) Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin. Nature 438:1176–1180

    Article  CAS  PubMed  Google Scholar 

  12. Fischle W, Tseng BS, Dormann HL, Ueberheide BM, Garcia BA, Shabanowitz J et al (2005) Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation. Nature 438:1116–1122

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Research in A.V.’s laboratory is funded by the Canadian Institutes for Health Research (CIHR, grant MOP 125916). Research in H.W.’s laboratory is funded by grants from the Canadian Institutes of Health Research (MOP 123438), and Natural Sciences and Engineering Research Council of Canada (RGPIN 435636-2013).

Author information

Authors and Affiliations

  1. Department of Biology, Université de Sherbrooke, Sherbrooke, QC, Canada, J1K 2R1

    Benoit Guillemette

  2. Centre de Recherche de l’Hôpital Maisonneuve-Rosemont, Montréal, QC, Canada, H1T 2M4

    Ian Hammond-Martel & Hugo Wurtele

  3. Département de Médecine, Université de Montréal, Montréal, QC, Canada, H3T 1J4

    Hugo Wurtele

  4. Institut de Recherche en Immunologie et Cancérologie (IRIC), Université de Montréal, CP 6128, Succursale Centre-Ville, Montréal, QC, Canada, H3C 3J7

    Alain Verreault

  5. Département de Pathologie et Biologie Cellulaire, Université de Montréal, CP 6128, Succursale Centre-Ville, Montréal, QC, Canada, H3C 3J7

    Alain Verreault

Authors
  1. Benoit Guillemette
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ian Hammond-Martel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hugo Wurtele
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alain Verreault
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alain Verreault .

Editor information

Editors and Affiliations

  1. Université de Sherbrooke Department of Biology, Sherbrooke, Québec, Canada

    Benoit Guillemette

  2. Department of Biology, Université de Sherbrooke, Sherbrooke, Québec, Canada

    Luc R. Gaudreau

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media New York

About this protocol

Cite this protocol

Guillemette, B., Hammond-Martel, I., Wurtele, H., Verreault, A. (2017). Production and Purification of Antibodies Against Histone Modifications. In: Guillemette, B., Gaudreau, L. (eds) Histones. Methods in Molecular Biology, vol 1528. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6630-1_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6630-1_9

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6628-8

  • Online ISBN: 978-1-4939-6630-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation