Two-step cross-linking for analysis of protein-chromatin interactions

Bing Tian, Jun Yang, Allan R. Brasier

Research output: Chapter in Book/Report/Conference proceedingChapter

89 Scopus citations


Eukaryotic gene regulation is controlled, in part, by inducible transcription factor-binding regulatory sequences in a tissue-specific and hormone-responsive manner. The development of methods for the analysis of transcription factor interaction within native chromatin has been a significant advance for the systematic analyses of the timing of gene regulation and studies on the effects of chromatin modifying enzymes on promoter accessibility. Chromatin immunoprecipitation (ChIP) is a specific method involving formaldehyde mediated protein-chromatin fixation to preserve the interaction for subsequent target identification. However, the conventional single-step cross-linking technique does not preserve all protein-DNA interactions, especially for transcription factors in hyper-dynamic equilibrium with chromatin or for coactivator interactions. Here, we describe a versatile, efficient "two-step" XChIP method that involves sequential protein-protein fixation followed by protein-DNA fixation. This method has been used successfully for analysis of chromatin binding for transcription factors (NF-κB, STAT3), polymerases (RNA Pol II), coactivators (CBP/p300, CDK9), and chromatin structural proteins (modified histones). Modifications of DNA extraction and sonication suitable for downstream target identification by quantitative genomic PCR and next generation sequencing are described.

Original languageEnglish (US)
Title of host publicationTranscriptional Regulation
Subtitle of host publicationMethods and Protocols
EditorsAles Vancura
Number of pages16
StatePublished - 2012
Externally publishedYes

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745


  • Chromatin immunoprecipitation
  • Next generation sequencing
  • Nuclear factor-κB
  • Polymerase chain reaction

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics


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