Isolation of intracellular protein--DNA complexes using HaloCHIP, an antibody-free alternative to chromatin immunoprecipitation

Methods Mol Biol. 2013:977:111-24. doi: 10.1007/978-1-62703-284-1_9.

Abstract

Mapping of protein binding sites within the genome has been significantly advanced by microarray and sequencing technologies, yet the method traditionally used to isolate protein-DNA complexes, chromatin immunoprecipitation, has remained dependent of the use of antibodies. Furthermore, cross-linking is commonly used to trap protein-DNA complexes and the challenge of using antibodies has come in recognition of the cross-linked epitopes, sometimes limiting the success of the approach. Here we present a method, HaloCHIP, which utilizes a HaloTag protein fusion and corresponding interaction resin, HaloLink, for capture of cross-linked protein-DNA complexes directly from a cellular lysate. This process alleviates the need for using an antibody, yields the DNA fragments bound to a particular protein of interest, and allows for a variety of downstream analyses such as PCR, qPCR, microarrays, and sequencing.

MeSH terms

  • Animals
  • Cell Culture Techniques
  • Cells, Cultured
  • Chromatin Immunoprecipitation / methods*
  • Cross-Linking Reagents / chemistry
  • DNA / isolation & purification*
  • DNA Cleavage
  • DNA-Binding Proteins / isolation & purification*
  • Formaldehyde / chemistry
  • Humans
  • Protein Binding
  • Sonication
  • Tissue Fixation

Substances

  • Cross-Linking Reagents
  • DNA-Binding Proteins
  • Formaldehyde
  • DNA