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


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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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.

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Authors and Affiliations



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.

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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).

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