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

ChIP assays of Col1a1, Ctgf, Pai1, and Acta2 were performed using a ChIP assay kit (Millipore, Temecula, CA) as previously described67. The resulting solutions from NRK-52E cells were incubated overnight at 4 °C with anti-histone H3.3 antibody (Abcam) or anti-HIRA antibody (Abcam). DNA was purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA). DNA samples were subjected to qPCR using gene-specific primers as follows:68 forward 5′-GGCTGGAGAAAGGTGGGTCT-3′ and reverse 5′-CCCAGGTATGCAGGGTAGGA-3′ were used for Col1a1, forward 5′-ATCAGGAAGGGTGCGAAGAG-3′ and reverse 5′-TCCACATTCCTCCGTCTGAA-3′ were used for Ctgf, forward 5′-gacaatATGTGCCCTGTGATTGtC-3′ and reverse 5′-AGGCTGCTCTACTGGTCCTTGC-3′ were used for Pai1 as described previously32, and forward 5′-GTAACATAGGCTGGTGAATCCTG-3′ and reverse 5′-GAGTGATCATTTTATGGAAGCCAC-3′ were used for Acta2 as described previously69.

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Manufacturer protocol

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

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