Chromatin Affinity Purification

  • Ryoko Harada
  • Alain NepveuEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 809)


Chromatin immunoprecipitation (ChIP) has become an essential assay in the field of transcriptional regulation. It is currently the most popular method to monitor the in vivo interaction between a protein and specific genomic sites. The method can also serve to identify novel transcriptional targets when the immunoprecipitated chromatin, sometimes called chipped DNA, is used either as a probe in hybridization experiments with microarrays of genomic DNA (ChIP-chip) or as template in DNA sequencing (ChIP-Seq). ChIP assays rely on the availability of good antibodies that can specifically and efficiently immunoprecipitate the protein under study even after cross-linking. However, good antibodies are not always available. To circumvent this problem, we have developed and validated the method of chromatin affinity purification (ChAP). The subsequent microarray analysis is then referred to as ChAP-chip. In brief, the protein under study is expressed together with two tags in order to allow the purification of chromatin by tandem affinity purification. To ensure that only true targets are identified, it is important to express the recombinant tagged-protein at physiological level. This requirement is not trivial as most expression vectors are designed to express proteins at high levels. We found most convenient to use an inducible retroviral vector in the absence of inducer and transactivator protein. We describe the procedure to generate cells stably expressing recombinant tagged-proteins at physiological level and then to purify the associated chromatin by affinity purification. Targets identified in this manner were validated in independent ChAP assays as well as in ChIP assays using antibodies against the endogenous protein.

Key words

Chromatin immunoprecipitation Affinity chromatography Tandem affinity purification Transcription factors Promoter Gene regulation Genome Genomic microarray 



We acknowledge the expert technical assistance of Ms. Ginette Bérubé and Mr. Lam Leduy in the preparation of retroviral vectors. This research was supported by grant #019389 from the Canadian Cancer Society to A.Nepveu.


  1. 1.
    Harada, R., Vadnais, C., Sansregret, L., Leduy, L., Berube, G., Robert, F., and Nepveu, A. (2008) Genome-wide location analysis and expression studies reveal a role for p110 CUX1 in the activation of DNA replication genes, Nucleic Acids Res 36, 189–202.Google Scholar
  2. 2.
    Sansregret, L., and Nepveu, A. (2008) The multiple roles of CUX1: Insights from mouse models and cell-based assays, Gene 412, 84–94.Google Scholar
  3. 3.
    Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., and Seraphin, B. (1999) A generic protein purification method for protein complex characterization and proteome exploration, Nat Biotechnol 17, 1030–1032.Google Scholar
  4. 4.
    Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M., and Seraphin, B. (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification, Methods 24, 218–229.Google Scholar
  5. 5.
    Buck, M. J., and Lieb, J. D. (2004) ChIP-chip: considerations for the design, analysis, and application of genome-wide chromatin immunoprecipitation experiments, Genomics 83, 349–360.Google Scholar
  6. 6.
    Oberley, M. J., Tsao, J., Yau, P., and Farnham, P. J. (2004) High-throughput screening of chromatin immunoprecipitates using CpG-island microarrays, Methods Enzymol. 376, 315–334.Google Scholar
  7. 7.
    Rodriguez, B. A., and Huang, T. H. (2005) Tilling the chromatin landscape: emerging methods for the discovery and profiling of protein-DNA interactions, Biochem Cell Biol 83, 525–534.Google Scholar
  8. 8.
    Taneyhill, L. A., and Adams, M. S. (2008) Investigating regulatory factors and their DNA binding affinities through real time quantitative PCR (RT-QPCR) and chromatin immunoprecipitation (ChIP) assays, Methods Cell Biol 87, 367–389.Google Scholar
  9. 9.
    Geck, P., Maffini, M. V., Szelei, J., Sonnenschein, C., and Soto, A. M. (2000) Androgen-induced proliferative quiescence in prostate cancer cells: the role of AS3 as its mediator, Proc Natl Acad Sci USA 97, 10185–10190.Google Scholar
  10. 10.
    Saqr, H. E., Omran, O., Dasgupta, S., Yu, R. K., Oblinger, J. L., and Yates, A. J. (2006) Endogenous GD3 ganglioside induces apoptosis in U-1242 MG glioma cells, J Neurochem 96, 1301–1314.Google Scholar
  11. 11.
    Sipione, S., Rigamonti, D., Valenza, M., Zuccato, C., Conti, L., Pritchard, J., Kooperberg, C., Olson, J. M., and Cattaneo, E. (2002) Early transcriptional profiles in huntingtin-inducible striatal cells by microarray analyses, Hum Mol Genet 11, 1953–1965.Google Scholar
  12. 12.
    Carey, M. F., Peterson, C. L., and Smale, S. T. (2009) Chapter 8: Confirming the Functional Importance of a Protein–DNA Interaction, in Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.The Rosalind and Morris Goodman Cancer Research CentreMcGill UniversityMontrealCanada
  2. 2.Departments of Biochemistry, Oncology, and Medicine, The Rosalind and Morris Goodman Cancer Research CentreMcGill UniversityMontrealCanada

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