UNIT 17.12 Use of In Vivo Biotinylation for Chromatin Immunoprecipitation
Published Online: 1 JUN 2011
Copyright © 2011 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Cell Biology
How to Cite
Kulyyassov, A., Shoaib, M. and Ogryzko, V. 2011. Use of In Vivo Biotinylation for Chromatin Immunoprecipitation. Current Protocols in Cell Biology. 51:17.12:17.12.1–17.12.22.
- Published Online: 1 JUN 2011
- Published Print: JUN 2011
This unit describes a system for expression of biotinylated proteins in mammalian cells in vivo, and its application to chromatin immunoprecipitation (ChIP). The system is based on co-expression of the target protein fused to a short biotin acceptor domain, together with the biotinylating enzyme BirA from Escherichia coli. The superior strength of the biotin-avidin interaction in the modified ChIP protocol presented here allows one to employ more stringent washing conditions, resulting in a better signal/noise ratio. Methods for interpreting the data obtained from ChIP samples analyzed by qPCR, and methods for testing the efficiency of biotinylation using a streptavidin gel-shift are also presented. In addition, a complementary method, based on isothermal multiple strand displacement amplification (IMDA) of circular concatemers generated from the DNA fragments obtained after ChIP, is described. This method helps to decrease bias in DNA amplification and is useful for the analysis of complex mixtures of DNA fragments typically generated in miniscale ChIP experiments. Curr. Protoc. Cell Biol. 51:17.12.1-17.12.22. © 2011 by John Wiley & Sons, Inc.
- chromatin immunoprecipitation;
- biotinylation in vivo;
- amplification bias