Analysis of Protein Co‐Occupancy by Quantitative Sequential Chromatin Immunoprecipitation

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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 ), 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

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Materials

Basic Protocol 1:

Materials

100 mg/ml bovine serum albumin (BSA, Fraction V; Sigma) in water (store at −20°C)
500 µg/ml λ phage DNA (not sheared; New England Biolabs)
10 mg/ml E. coli tRNA in water (store at −20°C)
20 mg/ml glycogen, or Pellet Paint (Novagen) as DNA carrier

Additional reagents and equipment for ChIP (unit 21.3 , Basic Protocol), growth of Saccharomyces cerevisiae (units 13.1 & 13.2 ), extraction and purification of DNA (unit 2.1 ), and real‐time quantitative PCR (unit 21.3 , Alternate Protocol 2)

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Figures

Figure 21.8.1 Schematic depiction of the three possible outcomes of a SeqChIP between proteins A and B: complete co‐occupancy (top left), no co‐occupancy (top right), and partial co‐occupancy (bottom panel). Partial co‐occupancy can be further subdivided into two categories: A is required (bottom left) or not required (bottom right) for the binding of B.

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Literature Cited

Literature Cited
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