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

HEK-293T cells have been transiently transfected using calcium phosphate [17] and 2 μg of FLAG-HDAC5 (Addgene #32213) expression plasmid in a 10-cm dish. After 48 h, cells were harvested and processed for ChIP using the ChIP-IT Express Chromatin Immunoprecipitation Kit (ChIP-IT Express, Active Motif, Carlsbad, CA, USA) according to the manufacturer’s recommendations. In parallel, FLAG-HDAC5 expression was determined by Western blotting (data not shown). For immunoprecipitation, 2.5 μg anti-Flag antibody (Sigma, Cat.#F3165) was used. Antibodies against acetyl-histone H3 (Cell Signalling, Cat.#9715) and mouse IgG (PP64B, Upstate, Millipore) served as positive and negative controls, respectively. PCR was performed using JumpStart Taq (Sigma-Aldrich) and the following primers, specifically selected to cover a 2500-bp genomic region upstream of the transcription start site (TSS) of the CDX2 gene: 2000–2500 bp, F: 5′-CTTTCCATGGCTGGAGCACT-3′, R: 5′-CGCTGGCTAATTGTCCCTGT-3′; 1500–2000 bp, F: 5′-CATTCCCACCCCATCAGGTC-3′, R: 5′-CCAAGGAGCTGTGCACTCAA-3′; 1000–1500 bp, F: 5′-ACAGACAAGTGCAGGTCTCC-3′, R: 5′-CCCAGCTCGGTTTCAGCA-3′; and TSS–500 bp, F: 5′-TGGAGGTTAAAGTGCACCAGGT-3′, R: 5′-GACACCAATGGTTGGAGACG-3′. As a positive control for HDAC5 recruitment, we amplified a genomic region of the HDAC5 repressed fibroblast growth factor 2 (FGF2) gene using the following published primers: F: 5′-TGGAGGTTAAAGTGCACCAGGT-3′ and R: 5′-GACACCAATGGTTGGAGACG-3′.

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Manufacturer protocol

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

Download manufacturer protocol

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