Chromatin Immunoprecipitation Assay (ChIP)
Chromatin Immunoprecipitation Assay, also known as the ChIP assay allows one to determine the mechanism of trascriptional repression, by immunoprecipitating chromatin with antibodies. ChIP involves formaldehyde treatment of a cell population to cross-link proteins to their target DNA in vivo, cell lysis, sonication to fragment chromatin, and then immunoprecipitation of specific chromatin complexes using an antibody against the protein of interest. After removal of the cross-links, PCR amplification at a selected locus, or slot blot hybridization using a specific probe is performed.
Chromatin Immunoprecipitation Assay / ChIP Advantages
Chromatin Immunoprecipitation Assay / ChIP has the advantage of measuring protein DNA interactions of promoter regions with protein complexes in the natural genomic state. Moreover, the ChIP assay can directly provide information on the histone acetylase status of specific chromatin regions, and be used to study non-histone proteins, even ones that do not directly bind DNA. It also minimizes the chances of chromatin rearrangements during preparation and precipitation, and it can be used with gene-specific primers for increased sensitivity or with a specific probe, slot blot hybridization, for increased specificity.
Chromatin Immunoprecipitation Assay / ChIP Disadvantages
Dificulties with using ChIp will include, the danger that the cross-linking step may fix interactions that are of minor functional sugnificance, precipitation us often very inefficient, and purifying cross-linked DNA-protein usually requires isopycnic centrifugation, a prolonged and expensive procedure. A large number of cells are usually required and the data represents an average of potentially multiple functional states in the cell population. In addition, negative results do not necessarily mean that a given factor is not associated with the site. Certain antibodies are poor in immunoprecipitating crosslinked chromatin and lack of crosslinking can results from inaccessibility of factors in large complexes or biochemical properties of proteins that decrease cross-linking efficiency.
Chromatin Immunoprecipitation Assay / ChIP Success
The likelihood of success with the Chromatin Immunoprecipitation Assay / ChIP method is protein and antibody dependent, but the methods track record suggests that this technique is extremely versatile and widely applicable. Success will depend on the abundance of the desired protein-DNA adducts being studied, the quality and affinity of the antibodies being used, and on the size and complexity of the genetic loci studied.
Alternative Approaches Instead of Chromatin Immunoprecipitation Assay / ChIP
Alternative approaches include, studies using conventional methods such as transient transfection assays, electrophoretic mobility shift assays (EMSA) and coimmunoprecipitation.
Beatrice N et al. Location analysis of DNA-bound proteins at the whole genome level: untangling transcriptional regulatory networks. Bioassays 23, 472-476 (2001)
Weinmann AS et al. Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation. Methods 26, 37-47 (2002)
Johnson KD et al. Dissecting long-range transcriptional mechanisms by chromatin immunoprecipitation. Methods 26, 27-36 (2002)
Kuo MH et al. In vivo cross-linking and immunoprecipitation for studying dynamic protein:DNA associations in a chromatin environment. Methods 19, 425-433 (1999)