ChIP-IT® Express Chromatin Immunoprecipitation Kits

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Do not re-freeze the Protein G magnetic beads, the beads should be stored at 4C.	-Always use ChIP validated antibody. -Optimal use of 1-3 ug of antibody.	-The diluted Proteinase K stop solution can not be stored.

Upstream tips
-Do not re-freeze the Protein G magnetic beads, the beads should be stored at 4C.
Protocol tips
-Always use ChIP validated antibody. -Optimal use of 1-3 ug of antibody.
Downstream tips
-The diluted Proteinase K stop solution can not be stored.

Publication protocol

ChiP assay was performed with the ChIP-IT™ Express Enzymatic Kit (Active Motif, CA, USA), following the manufacturer's guidelines. THP-1 cells were cultured for 24 hours with methyl donors and were then differentiated with TPA (25 ng/μl) for 48 hours and activated with LPS (100 ng/μl) over 24 hours. The cell medium was discarded and 36.5% formaldehyde was added directly to the cell surface for 10 min. Cross-linking between proteins and DNA was stopped by the addition of glycine for 5 min at room temperature and cells were collected by scraping. Then, cells were incubated with lysis buffer for 30 min at 4°C and DNA was fragmented via enzyme-based digestion for 10 min at 37°C. Chromatin was immunoprecipitated using rabbit polyclonal antibody to nuclear factor-κB (NF-κB; ab7970, Abcam, MA, USA). After immunoprecipitation, cross-linking of protein-DNA complexes was reversed. Real-time quantitative PCR was performed using primers for IL1B: sense 5′-agcaacaaagctgccactta-3′ and antisense 5′-tgacgtgctgtgtgaatttg-3′, and TNF: sense 5′-ggagaatgtccagggctatg-3′ and antisense 5′-tcctggaggctctttcactc-3′.

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Papers

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Manufacturer protocol

Download the product protocol from Active Motif for ChIP-IT® Express Chromatin Immunoprecipitation Kits below.

Download manufacturer protocol

Videos

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