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

Chromatin immunoprecipitation (ChIP) was performed using an assay kit (Millipore) with minor modifications. Briefly, cells were fixed in 1% formaldehyde, then lysed in SDS buffer. Cells were then sonicated using a Branson Digital Sonifier 450 (VWR, West Chester, PA, USA) to create 200–1000 bp fragments. The lysate was clarified by centrifugation, and 5% of the supernatant was saved to measure the input DNA. The remaining chromatin was incubated with 1 μg of anti-H3K4me3 antibody (Abcam) or control IgG (Millipore) and incubated at 4°C with rotation overnight. Immune complexes were precipitated with salmon sperm DNA/protein A agarose beads. Crosslinking was reversed by incubation at 65°C and samples were treated with proteinase K. DNA was purified by phenol:chloroform:isoamyl alcohol separation and ethanol precipitation. Primers for the promoter regions of IFN-β, TNF-α and IL-6 were designed using Lasergene software and DNA was amplified by qPCR using SYBR Green buffer (Applied Biosystems).

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Manufacturer protocol

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

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