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

F-NL cells were crosslinked using 1% formaldehyde after 72 h of treatment with SAHA in the presence or absence of TGF-β1 stimulation. ChIP assays were performed using the ChIP-IT Express Kit (53008, Active Motif, Carlsbad, CA, USA) as described previously [5]. Antibodies against acetylated histone H3 (06-599, Merck Millipore, Billerica, MA, USA), H3K27me3 (07-499, Merck Millipore), or normal rabbit IgG (12-370, Merck Millipore) were used for immunoprecipitation. 5 μl of purified immunoprecipitated-DNA was amplified using real-time PCR amplification with primers (0.5 μM) designed specifically for the COX-2 promoter region (set A and B, Fig. 2A) and the KAPA SYBR® FAST qPCR Kit (KK4602, Roche Diagnostics). The primer sequences were the following: Set A forward 5′-ACAGCCTATTAAGCGTCGTCA-3′ and reverse 5′-CCGTGTCTGGTCTGTACGTC-3′, Set B forward 5′-AGCTTCCTGGGTTTCCGATT-3′ and reverse 5′-AGCCCATGTGACGAAATGACT-3′. ChIP data were analyzed using the Percent Input (% Input) method. Briefly, input Ct values corresponding to 1% of initial chromatin were adjusted to 100% and used to normalize Ct values of immunoprecipitated-DNA samples. ChIP PCR was performed in duplicate and the results were presented as mean ± SEM of three independent biological replicates (cell lines).

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Manufacturer protocol

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

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