Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Protocol for the fast chromatin immunoprecipitation (ChIP) method

Abstract

Chromatin and transcriptional processes are among the most intensively studied fields of biology today. The introduction of chromatin immunoprecipitations (ChIP) represents a major advancement in this area. This powerful method allows researchers to probe specific protein-DNA interactions in vivo and to estimate the density of proteins at specific sites genome-wide. We have introduced several improvements to the traditional ChIP assay, which simplify the procedure, greatly reducing the time and labor required to complete the assay. The simplicity of the method yields highly reproducible results. Our improvements facilitate the probing of multiple proteins in a single experiment, which allows for the simultaneous monitoring of many genomic events. This method is particularly useful in kinetic studies where multiple samples are processed at the same time. Starting with sheared chromatin, PCR-ready DNA can be isolated from 16–24 ChIP samples in 4–6 h using the fast method.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: General outline of the fast ChIP procedure.
Figure 2: Short incubation of chromatin with antibodies in ultrasonic bath is equivalent to overnight binding.
Figure 3: Example of fast ChIP assay done with multiple antibodies in a single experiment.
Figure 4: Timeline of the fast ChIP protocol.

Similar content being viewed by others

References

  1. Bernstein, E. & Allis, C.D. RNA meets chromatin. Genes Dev. 19, 1635–1655 (2005).

    Article  CAS  PubMed  Google Scholar 

  2. Schubeler, D. & Elgin, S.C. Defining epigenetic states through chromatin and RNA. Nat. Genet. 37, 917–918 (2005).

    Article  PubMed  Google Scholar 

  3. Felsenfeld, G. & Groudine, M. Controlling the double helix. Nature 421, 448–453 (2003).

    Article  PubMed  Google Scholar 

  4. Sims, R.J. III, Mandal, S.S. & Reinberg, D. Recent highlights of RNA-polymerase-II-mediated transcription. Curr. Opin. Cell Biol. 16, 263–271 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. Thiriet, C. & Hayes, J.J. Chromatin in need of a fix: phosphorylation of H2AX connects chromatin to DNA repair. Mol. Cell 18, 617–622 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Kuo, M.H. & Allis, C.D. In vivo cross-linking and immunoprecipitation for studying dynamic Protein: DNA associations in a chromatin environment. Methods 19, 425–433 (1999).

    Article  CAS  PubMed  Google Scholar 

  7. Orlando, V., Strutt, H. & Paro, R. Analysis of chromatin structure by in vivo formaldehyde cross-linking. Methods 11, 205–214 (1997).

    Article  CAS  PubMed  Google Scholar 

  8. Impey, S. et al. Defining the CREB regulon: a genome-wide analysis of transcription factor regulatory regions. Cell 119, 1041–1054 (2004).

    CAS  PubMed  Google Scholar 

  9. Nelson, J.D., Denisenko, O., Sova, P. & Bomsztyk, K. Fast chromatin immunoprecipitation assay. Nucleic Acids Res. 34, e2 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Ostrowski, J., Kawata, Y., Schullery, D.S., Denisenko, O.N. & Bomsztyk, K. Transient recruitment of the hnRNP K protein to inducibly transcribed gene loci. Nucleic Acids Res. 31, 3954–3962 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Dundr, M. et al. A kinetic framework for a mammalian RNA polymerase in vivo. Science 298, 1623–1626 (2002).

    Article  CAS  PubMed  Google Scholar 

  12. Cheutin, T. et al. Maintenance of stable heterochromatin domains by dynamic HP1 binding. Science 299, 721–725 (2003).

    Article  CAS  PubMed  Google Scholar 

  13. Cosma, M.P., Tanaka, T. & Nasmyth, K. Ordered recruitment of transcription and chromatin remodeling factors to a cell cycle- and developmentally regulated promoter. Cell 97, 299–311 (1999).

    Article  CAS  PubMed  Google Scholar 

  14. Liu, C.L. et al. Single-nucleosome mapping of histone modifications in S. cerevisiae. PLoS Biol. 3, e328 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Metivier, R. et al. Transcriptional complexes engaged by apo-estrogen receptor-alpha isoforms have divergent outcomes. EMBO J. 23, 3653–3666 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Koyanagi, M. et al. EZH2 and histone 3 trimethyl lysine 27 associated with Il4 and Il13 gene silencing in Th1 cells. J. Biol. Chem. 280, 31470–31477 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Solomon, M.J. & Varshavsky, A. Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures. Proc. Natl. Acad. Sci. USA 82, 6470–6474 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Thorne, A.W., Myers, F.A. & Hebbes, T.R. Native chromatin immunoprecipitation. Methods Mol. Biol. 287, 21–44 (2004).

    CAS  PubMed  Google Scholar 

  19. Bernstein, B.E. et al. Genomic maps and comparative analysis of histone modifications in human and mouse. Cell 120, 169–181 (2005).

    Article  CAS  PubMed  Google Scholar 

  20. Pokholok, D.K. et al. Genome-wide map of nucleosome acetylation and methylation in yeast. Cell 122, 517–527 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Cawley, S. et al. Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs. Cell 116, 499–509 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Orlando, V. Mapping chromosomal proteins in vivo by formaldehyde-crosslinked- chromatin immunoprecipitation. Trends Biochem. Sci. 25, 99–104 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Chen, R. et al. Ultrasound-accelerated immunoassay, as exemplified by enzyme immunoassay of choriogonadotropin. Clin. Chem. 30, 1446–1451 (1984).

    CAS  PubMed  Google Scholar 

  24. Chaya, D. & Zaret, K.S. Sequential chromatin immunoprecipitation from animal tissues. Methods Enzymol. 376, 361–372 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Schwabish, M.A. & Struhl, K. Evidence for eviction and rapid deposition of histones upon transcriptional elongation by RNA polymerase II. Mol. Cell. Biol. 24, 10111–10117 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Denisenko, O. & Bomsztyk, K. Yeast hnRNP K-like genes are involved in regulation of the telomeric position effect and telomere length. Mol. Cell. Biol. 22, 286–297 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kurdistani, S.K., Tavazoie, S. & Grunstein, M. Mapping global histone acetylation patterns to gene expression. Cell 117, 721–733 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Sandoval, J. et al. RNAPol-ChIP: a novel application of chromatin immunoprecipitation to the analysis of real-time gene transcription. Nucleic Acids Res. 32, e88 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Waugh, D.S. Making the most of affinity tags. Trends Biotechnol. 23, 316–320 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Van Seuningen, I., Ostrowski, J. & Bomsztyk, K. Description of an IL-1-responsive kinase that phosphorylates the K protein. Enhancement of phosphorylation by sequence-selective DNA and RNA motifs. Biochemistry 34, 5644–5650 (1995).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank members of the K.B. lab for valuable discussions of the method. This work was supported by the US National Institutes of Health (DK45978 and GM45134) and the Juvenile Diabetes Research Foundation (K.B.).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Karol Bomsztyk.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nelson, J., Denisenko, O. & Bomsztyk, K. Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nat Protoc 1, 179–185 (2006). https://doi.org/10.1038/nprot.2006.27

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.27

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing