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Epitope tagging of endogenous proteins for genome-wide ChIP-chip studies

Abstract

We developed a strategy to introduce epitope tag–encoding DNA into endogenous loci by homologous recombination–mediated 'knock-in'. The tagging method is straightforward, can be applied to many loci and several human somatic cell lines, and can facilitate many functional analyses including western blot, immunoprecipitation, immunofluorescence and chromatin immunoprecipitation–microarray (ChIP-chip). The knock-in approach provides a general solution for the study of proteins to which antibodies are substandard or not available.

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Figure 1: Schematic diagram of tagging endogenous protein with 3 × Flag.
Figure 2: 3 × Flag tagged proteins are detectable by western blot, immunoprecipitation and immunofluorescence.
Figure 3: ChIP analysis of wild-type and Flag-tagged STAT3.

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References

  1. Zhang, X. et al. Proc. Natl. Acad. Sci. USA 104, 4060–4064 (2007).

    Article  CAS  Google Scholar 

  2. Cherry, S.M. et al. Curr. Biol. 17, 373–378 (2007).

    Article  CAS  Google Scholar 

  3. The ENCODE Project Consortium. Science 306, 636–640 (2004).

  4. Scacheri, P.C., Crawford, G.E. & Davis, S. Methods Enzymol. 411, 270–282 (2006).

    Article  CAS  Google Scholar 

  5. Scacheri, P.C. et al. PLoS Genet. 2, e51 (2006).

    Article  Google Scholar 

  6. Frith, M.C. et al. Nucleic Acids Res. 32, 1372–1381 (2004).

    Article  CAS  Google Scholar 

  7. Wingender, E., Dietze, P., Karas, H. & Knuppel, R. Nucleic Acids Res. 24, 238–241 (1996).

    Article  CAS  Google Scholar 

  8. Ginsberg, M. et al. Mol. Cell. Biol. 27, 6300–6308 (2007).

    Article  CAS  Google Scholar 

  9. Waris, G. & Siddiqui, A. J. Virol. 76, 2721–2729 (2002).

    Article  CAS  Google Scholar 

  10. Bitinaite, J. et al. Nucleic Acids Res. 35, 1992–2002 (2007).

    Article  CAS  Google Scholar 

  11. Johnson, D.S., Mortazavi, A., Myers, R.M. & Wold, B. Science 316, 1497–1502 (2007).

    Article  CAS  Google Scholar 

  12. Lee, T.I. et al. Science 298, 799–804 (2002).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank D. Sedwick for helpful discussions, J. Yu for technical assistance, and P. Harte and G. Crawford for critically reading this manuscript. This work was supported by grants from US National Institutes of Health (CA127590, U54CA116867), Concern Foundation and V foundation to Z. Wang, and National Institutes of Health grants KCA103843A and RHD056369A to P.C.S.

Author information

Authors and Affiliations

Authors

Contributions

Z. Wang conceived and designed the experiments. X.Z. developed the tagging strategy and modified the targeting vector for uracil-specific excision reagent (USER) compatible cloning. C.G. performed experiments on the N protein. Y.C. tagged the MRE11 and PTPN14 proteins. H.P.S. and Z. Weng performed the motif identification analyses. P.C.S., M.P.S. and C.F.B. performed all ChIP analyses. T.L. analyzed ChIP data. P.C.S., Z. Wang and S.M. wrote the paper.

Note: Supplementary information is available on the Nature Methods website.

Corresponding authors

Correspondence to Peter C Scacheri or Zhenghe Wang.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1–2, Supplementary Methods (PDF 611 kb)

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Zhang, X., Guo, C., Chen, Y. et al. Epitope tagging of endogenous proteins for genome-wide ChIP-chip studies. Nat Methods 5, 163–165 (2008). https://doi.org/10.1038/nmeth1170

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