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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Zhang, X. et al. Proc. Natl. Acad. Sci. USA 104, 4060–4064 (2007).
Cherry, S.M. et al. Curr. Biol. 17, 373–378 (2007).
The ENCODE Project Consortium. Science 306, 636–640 (2004).
Scacheri, P.C., Crawford, G.E. & Davis, S. Methods Enzymol. 411, 270–282 (2006).
Scacheri, P.C. et al. PLoS Genet. 2, e51 (2006).
Frith, M.C. et al. Nucleic Acids Res. 32, 1372–1381 (2004).
Wingender, E., Dietze, P., Karas, H. & Knuppel, R. Nucleic Acids Res. 24, 238–241 (1996).
Ginsberg, M. et al. Mol. Cell. Biol. 27, 6300–6308 (2007).
Waris, G. & Siddiqui, A. J. Virol. 76, 2721–2729 (2002).
Bitinaite, J. et al. Nucleic Acids Res. 35, 1992–2002 (2007).
Johnson, D.S., Mortazavi, A., Myers, R.M. & Wold, B. Science 316, 1497–1502 (2007).
Lee, T.I. et al. Science 298, 799–804 (2002).
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
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
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–5, Supplementary Tables 1–2, Supplementary Methods (PDF 611 kb)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nmeth1170
This article is cited by
-
Phosphorylation at tyrosine 317 and 508 are crucial for PIK3CA/p110α to promote CRC tumorigenesis
Cell & Bioscience (2023)
-
Nuclear translocation of p85β promotes tumorigenesis of PIK3CA helical domain mutant cancer
Nature Communications (2022)
-
P53-R273H mutation enhances colorectal cancer stemness through regulating specific lncRNAs
Journal of Experimental & Clinical Cancer Research (2019)
-
UBN1/2 of HIRA complex is responsible for recognition and deposition of H3.3 at cis-regulatory elements of genes in mouse ES cells
BMC Biology (2018)
-
Essential role of insulin-like growth factor 2 in resistance to histone deacetylase inhibitors
Oncogene (2016)