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

ChIP assays were performed as previously described [22]. The EZ-magna ChIP kit (Millipore, Bedford, MA, USA) was used according to the manufacturer’s protocol. Crosslinked chromatin was sonicated into DNA fragments in the range of 200–1000 bp and immunoprecipitated using rabbit anti-p-STAT3 antibodies (Abcam). Negative control samples were prepared using control rabbit anti-IgG antibody (Abcam), and rabbit anti-RNA Polymerase II antibody (Abcam) was used for positive control. After immunoprecipitation, the beads were washed sequentially with low-salt buffer, high-salt buffer, LiCl buffer, and TE buffer each for 5 min at 4 °C. The immunoprecipitated DNA was then eluted by incubation in 100 μl of elution buffer (0.1 M NaHCO3 and 1% SDS) containing 10 μg proteinase K (Millipore) at 62 °C for 2 h with rotation. The eluted DNA was purified using the columns and buffers in the kit and then re-dissolved in 50 μl of PCR-grade water. Immunoprecipitated chromatin was analyzed by qPCR using primers targeting the phosphorylated (p)-STAT3 (p-STAT3) binding regions in the human miR155HG promoter. The primer sequences used for ChIP-qPCR are binding region 1 (− 1982 to − 1972): F 5′-GAGACATCATTATTGTCATT-3′, R 5′-TAGGAGTCAAATACACCTG-3′; binding region 2 (− 1548 to − 1411): F 5′-ATGGGAAATTCAGAAAGGC-3′, R 5′-TGATCATATGAGGGAGGAGC-3′; and binding region 3 (− 275 to − 116): F 5′-TTAAGAACAAAGGTTGGAGC-3′, R 5′-TGTGACTCATAACCGACCAG-3′. PCR conditions were set according to the instructions provided in the SYBR Green Kit (Roche Applied Science, Upper Bavaria, Germany). Results were analyzed by agarose gel electrophoresis.

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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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