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

Article metrics

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1

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

  2. 2

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

  3. 3

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

  4. 4

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

  5. 5

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

  6. 6

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

  7. 7

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

  8. 8

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

  9. 9

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

  10. 10

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

  11. 11

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

  12. 12

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

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

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.

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)

Rights and permissions

Reprints and Permissions

About this article

Further reading