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Chromatin Immunoprecipitation Assay (ChIP)

Chromatin immunoprecipitation assay is an antibody-based technology used to investigate the interaction between proteins and DNA in the cell. Chip assays monitor transcriptional regulation and identify linkages between the genome and proteome through histone modifications (epigenetics) or transcription factor-DNA binding interactions.

Cross-link DNA and proteins in cells under physiological conditions cut chromatin into small fragments by sonication or enzymatic treatment. Using the specific recognition reaction of antigen and antibody, the DNA fragments bound to the target protein are precipitated to enrich the DNA fragments with histone modification or transcriptional regulation. The DNA sequence of this enriched fragment is then detected by a variety of downstream detection techniques (quantitative PCR, gene chip, sequencing, etc.).

Procedure
  • Crosslinking
    Crosslink DNA and associated proteins on chromatin in living cells or tissues with formaldehyde or other crosslinking agents.
  • Cell lysis
    Dissolve cell membranes by a detergent solution to release cellular components and then bring protein-DNA complexes into solution.
  • Chromatin preparation (shearing/digestion)
    Shear the DNA-protein complexes (chromatin-protein) into ~500 bp DNA fragments by sonication or nuclease digestion.
  • Immunoprecipitation
    Bind the antibody against the protein of interest to the protein-DNA complex, and then precipitate the complex.
  • Decrosslinking and DNA purification
    Broke the cross-links between the protein and the DNA by heat incubation or by protease digestion.
  • DNA analysis
    Analyze the degree of enrichment of DNA via PCR, qPCR, microarray, or NGS.

The basic flow chart for ChIP. Fig. 1 The basic flow chart for ChIP.

Types
  • Cross-linked ChIP (XChIP)
    X-ChIP can be used with histone and non-histone proteins. It generally requires less cellular starting material than N-ChIP and minimizes the chances of chromatin protein loss during extraction to detect transient protein interactions. However, the precipitation step is inefficient and downstream analysis requires DNA amplification by PCR.
  • Native ChIP (NChIP)
    In N-ChIP, no fixing agent is used to crosslink proteins to the chromatin. Antibodies can be recognized and bound to their target antigens more effectively. However, it is only capable of detecting histones. In addition, protein binding may be lost during the chromatin digestion and immunoprecipitation steps, which may bias data or impede proper analyses.
Features
  • Screen and identify protein binding sites on the whole chromosome efficiently and accurately in combination with NGS.
  • Can be coupled to many commonly used molecular biology techniques.
Applications
  • For the analysis of proteins interacting within a native chromatin environment.
  • Determine the allele-specific transcription factor binding patterns and measure long-range enhancer binding of specific proteins.
  • Uncover an extraordinarily rich and dynamic chromatin environment.
  • Provide useful end-point measurement for delineation of components within the signal transduction pathways
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STEMart provides you with a variety of ChIP equipment or consumables to meet your various R&D and application needs. If you have any questions or requirements for ChIP, please feel free to contact us.

Reference

  • Gade P, Kalvakolanu DV. Chromatin immunoprecipitation assay as a tool for analyzing transcription factor activity. Methods Mol Biol. 2012;809:85-104. doi: 10.1007/978-1-61779-376-9_6. PMID: 22113270; PMCID: PMC3891665.

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