Unit

UNIT 21.8 Analysis of Protein Co-Occupancy by Quantitative Sequential Chromatin Immunoprecipitation

  1. Joseph V. Geisberg,
  2. Kevin Struhl

Published Online: 1 MAY 2005

DOI: 10.1002/0471142727.mb2108s70

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Geisberg, J. V. and Struhl, K. 2005. Analysis of Protein Co-Occupancy by Quantitative Sequential Chromatin Immunoprecipitation. Current Protocols in Molecular Biology. 70:21.8:21.8.1–21.8.7.

Author Information

  1. Harvard Medical School, Boston, Massachusetts

Publication History

  1. Published Online: 1 MAY 2005
  2. Published Print: APR 2005

Abstract

Sequential Chromatin Immunoprecipitation (SeqChIP) is a powerful technique for analyzing the simultaneous association of two different proteins with genomic DNA sequences in vivo. Cellular Protein-DNA complexes are cross-linked with formaldehyde (unit Unavailable ), and are purified via two successive immunoprecipitations, with each immunoprecipitation targeting a different protein. Protein-DNA cross-links are then reversed and DNA sequences of interest are analyzed by quantitative PCR. At each genomic region, calculated SeqChIP co-occupancy values are compared to occupancy values of singly immunoprecipitated samples. The extent of enrichment brought about by the second immunoprecipitation relative to the singly immunoprecipitated sample is directly correlated with the degree of co-occupancy between the two proteins at the genomic location assayed. In principle, the technique is not limited to Saccharomyces cerevisiae. Cells from a wide variety of organisms can be used.

Keywords:

  • chromatin;
  • immunoprecipitation;
  • protein-DNA interactions;
  • in vivo crosslinking