EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation 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 were performed using Magna ChIP kit (Millipore) according to manufacturer’s instructions with modifications. Briefly, THP-1 cells were cultured in 10-cm dishes, and experiments were conducted using 1´107 cells. After administration of THP-1 cells with RORa-specific ligand CPG 52608 (5 nM), ChIP was performed. After 24 hours of incubation, cells were treated with 1% formaldehyde (Merck). Cell lysates were sonicated on ice, 20 times for 10 s and separated by 20 s. Immunoprecipitations were performed by incubation of fragmented chromatin with appropriate antibodies overnight at 4°C. ChIP reactions contained the following antibodies: anti-mouse IgG (Millipore), anti-RNA Pol II (Millipore), and anti-RORa antibody (Santa Cruz Biotechnology). An aliquot of the cell lysates was used to isolate total input DNA. Amplifications of the immunoprecipitated DNA were performed using PCR. GAPDH gene was used as control for ChIP–PCR with primers 5¢-TACTAGCGGTTTTACGGGCG-3¢ (forward) and 5¢-TCGAACAGGAGGAGCAGAGAGCGA-3¢ (reverse).

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Manufacturer protocol

Download the product protocol from Merck Millipore for EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit below.

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