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

Chromatin immunoprecipitation was performed with the use of EZ-Magna ChIP A-Chromatin Immunoprecipitation Kits (Millipore). Briefly, mononuclear cells in peripheral blood samples were isolated using Dynabeads FlowComp Human CD14 Kits (ThermoFisher Scientific) and cross-linked with 1% formaldehyde for 10 minutes. Thereafter, cells were lysed on ice, and chromatin DNA was sheared by sonication into fragments of between 0.2 and 1.0 kilobases in length. The chromatin DNA-protein fractions were incubated with an anti-RNA polymerase II (Pol II) antibody (ab5131, Abcam) overnight at 4°C, followed by precipitation of the immunocomplexes using protein A beads. DNA in the immunocomplexes was then isolated and subjected to real-time PCR of the FURIN gene and the housekeeping gene GAPDH. The ΔΔCt method was used to ascertain differences between genotypes in RNA Pol II occupancy in the FURIN gene, standardized against the reference gene GAPDH.

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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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