Chromatin Immunoprecipitation (ChIP) Assay Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Just because an antibody works well in a Western blot does not always indicate it will perform well in chromatin immunoprecipitation. Unlike a Western blot that detects proteins that have been denatured, a ChIP antibody must recognize the target protein in its native state. Use ChIP-validated antibody. -Use 2-10 μg of your ChIP antibody depending on the abundance of your protein target.

Protocol tips
-Just because an antibody works well in a Western blot does not always indicate it will perform well in chromatin immunoprecipitation. Unlike a Western blot that detects proteins that have been denatured, a ChIP antibody must recognize the target protein in its native state. Use ChIP-validated antibody. -Use 2-10 μg of your ChIP antibody depending on the abundance of your protein target.

Publication protocol

To verify genes under the transcriptional control of Clock, a ChIP assay was carried out. The ChIP assay was performed as described previously 37. As the cells were grown at 80% confluency in a 10‐cm2 dish, the number of cells was about 5 × 106. The cells were harvested, and the ChIP assay was performed according to the protocol for EZ‐ChIP™ Chromatin Immunoprecipitation Kit (Millipore, Billerica, MA, USA). The cells were crosslinked with formaldehyde at a final concentration of 1% (w/v) for 10 min at room temperature. The formaldehyde was neutralized by 125 mM glycine. Subsequently, the cells were washed and collected in cold PBS‐containing Protease Inhibitor Cocktail II. Nuclear pellets were resuspended and sonicated in SDS lysis buffer. The crosslinked DNA was sheared into ∼200 to 1,000 bp fragments. Each sonicated chromatin fragment was divided into three immunoprecipitation (IP) reactions. Protein G agarose was added to samples to delete nonspecific proteins and incubated at 4 °C with rotation for 1 h. IP reaction was immunoprecipitated with 5 μg rabbit polyclonal anti‐CLOCK (Cell Signaling, Danvers, MA, USA) or 1 μg normal mouse IgG. Antibody complexes were obtained with protein G agarose. Then, the immunocomplexes were reverse‐crosslinked, and the immunoprecipitated DNA was separated from protein. Real‐time PCR was performed, and the primers are listed in Table 1. Relative enrichment of anti‐CLOCK antibody and normal IgG was analyzed. The ChIP assay was used to determine the transcriptional regulation of target genes.

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Manufacturer protocol

Download the product protocol from Merck Millipore for Chromatin Immunoprecipitation (ChIP) Assay Kit below.

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