Advertisement

The Fast Chromatin Immunoprecipitation Method

  • Joel Nelson
  • Oleg Denisenko
  • Karol Bomsztyk
Part of the Methods in Molecular Biology book series (MIMB, volume 567)

Abstract

The chromatin immunoprecipitation assay (ChIP assay) has greatly facilitated the recent, dramatic expansion of our knowledge of the protein–DNA interactions involved in regulating gene expression, DNA repair, and cell division. The power of the assay is that it gives a researcher the ability to not only detect a specific protein–DNA interaction in vivo but also determine the relative density of factors along genes or the entire genome. Though powerful, the traditional assay is time consuming (involving 2 days or more) and laborious. With Fast ChIP, we simplified the assay to greatly reduce the time and labor involved. The improved assay is especially useful for studies which involve many samples, including the probing of multiple chromatin factors simultaneously and/or looking at genomic events over several time points. Using Fast ChIP, 24 sheared chromatin samples can be processed to yield PCR-ready DNA in 5 h.

Key words

Chromatin immunoprecipitation ChIP-chip tissue ChIP transcription DNA repair 

Notes

Acknowledgment

We thank members of the KB lab for valuable discussions of the method. This work was supported by NIH DK45978 and GM45134 to K.B.

References

  1. 1.
    Bernstein, E. and Allis, C. D. (2005) RNA meets chromatin.Genes Dev. 19, 1635–1655.PubMedCrossRefGoogle Scholar
  2. 2.
    Schubeler, D. and Elgin, S. C. (2005) Defining epigenetic states through chromatin and RNA. Nat. Genet. 37, 917–918.PubMedCrossRefGoogle Scholar
  3. 3.
    Felsenfeld, G. and Groudine, M. (2003) Controlling the double helix. Nature 421, 448–453.PubMedCrossRefGoogle Scholar
  4. 4.
    Sims, R. J., 3rd, Mandal, S. S. and Reinberg, D. (2004) Recent highlights of RNA-polymerase-II-mediated transcription. Curr. Opin. Cell Biol. 16, 263–271.PubMedCrossRefGoogle Scholar
  5. 5.
    Thiriet, C. and Hayes, J. J. (2005) Chromatin in need of a fix: phosphorylation of H2AX connects chromatin to DNA repair. Mol. Cell 18, 617–622.PubMedCrossRefGoogle Scholar
  6. 6.
    Kuo, M. H. and Allis, C. D. (1999) In vivo cross-linking and immunoprecipitation for studying dynamic protein:DNA associations in a chromatin environment. Methods 19, 425–433.PubMedCrossRefGoogle Scholar
  7. 7.
    Orlando, V., Strutt, H. and Paro, R. (1997) Analysis of chromatin structure by in vivo formaldehyde cross-linking. Methods 11, 205–214.PubMedCrossRefGoogle Scholar
  8. 8.
    Solomon, M. J. and Varshavsky, A. (1985) Formaldehyde-mediated DNA–protein crosslinking: a probe for in vivo chromatin structures. Proc. Natl. Acad. Sci. U.S.A. 82, 6470–6474.PubMedCrossRefGoogle Scholar
  9. 9.
    Thorne, A. W., Myers, F. A. and Hebbes, T. R. (2004) Native chromatin immunoprecipitation. Methods Mol. Biol. 287, 21–44.PubMedGoogle Scholar
  10. 10.
    Solomon, M. J., Larsen, P. L. and Varshavsky, A. (1988) Mapping protein–DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell 53, 937–947.PubMedCrossRefGoogle Scholar
  11. 11.
    Nelson, J. D., Denisenko, O., Sova, P. and Bomsztyk, K. (2006) Fast chromatin immunoprecipitation assay. Nucleic Acids Res. 34, e2.PubMedCrossRefGoogle Scholar
  12. 12.
    Huebert, D. J., Kamal, M., O'Donovan, A. and Bernstein, B. E. (2006) Genome-wide analysis of histone modifications by ChIP-on-chip. Methods 40, 365–369.PubMedCrossRefGoogle Scholar
  13. 13.
    Johnson, D. S., Mortazavi, A., Myers, R. M. and Wold, B. (2007) Genome-wide mapping of in vivo protein–DNA interactions. Science 316, 1497–1502.PubMedCrossRefGoogle Scholar
  14. 14.
    Chen, R., Weng, L., Sizto, N. C., Osorio, B., Hsu, C. J., Rodgers, R. and Litman, D. J. (1984) Ultrasound-accelerated immunoassay, as exemplified by enzyme immunoassay of choriogonadotropin. Clin. Chem. 30, 1446–1451.PubMedGoogle Scholar
  15. 15.
    Nelson, J. D., Flanagin, S., Kawata, Y., Denisenko, O. and Bomsztyk, K. (2008) Transcription of laminin {gamma}1 chain gene in rat mesangial cells: constitutive and inducible RNA polymerase II recruitment and chromatin states. Am. J. Physiol. Renal. Physiol. 294, F525–533.Google Scholar
  16. 16.
    Zager, R. A., Johnson, A. C., Naito, M. and Bomsztyk, K. (2008) Maleate nephrotoxicity: mechanisms of injury and correlates with ischemic/hypoxic tubular cell death. Am. J. Physiol. Renal. Physiol. 294, F187–197.Google Scholar
  17. 17.
    Denisenko, O. and Bomsztyk, K. (2008) Epistatic interaction between the K-homology domain protein HEK2 and SIR1 at HMR and telomeres in yeast. J. Mol. Biol. 375, 1178–1187.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Joel Nelson
    • 1
  • Oleg Denisenko
    • 2
  • Karol Bomsztyk
    • 1
    • 2
  1. 1.Molecular and Cellular Biology ProgramUniversity of WashingtonSeattleUSA
  2. 2.UW Medicine at Lake UnionUniversity of WashingtonSeattleUSA

Personalised recommendations