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

The method of purification of rat megakaryocytes has been described previously.11 Purified megakaryocytes were incubated for 2 hours in IMDM containing 5% FBS, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 0.2 ng/mL thrombopoietin (TPO; donated by Kirin Brewery). After the incubation, the ChIP assay was performed using a Chromatin Immunoprecipitation Assay Kit (Upstate, Lake Placid, NY) according to the manufacturer's instructions. For the assay, 10 μg of antibodies for USF1 and 2 described above or subtype-matched normal rabbit IgG (Santa Cruz Biotechnology) was used. The PCR amplification for the fragment including the TME was performed by 35 cycles of 3 steps (94° C for 30 seconds, 55° C for 30 seconds, and 72° C for 30 seconds) using the primers, forward 5′-CATACAGCATACCTTCTGCG-3′ and reverse 5′-AAGCAGCTGAGGCCAGCTGTCAGCA-3′. The PCR amplification for hypoxanthine phosphoribosyltransferase (Hprt) was performed by 40 cycles of 3 steps (94° C for 30 seconds, 60° C for 30 seconds, and 72° C for 30 seconds) using the primers, forward 5′-GGAGATTGAGGGAGAGCAAACTCAG-3′ and reverse 5′-CTCTTCCCACTGATGTCCTACAAGG-3′. PCR products were separated on a 2% agarose gel and stained by ethidium bromide.

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Manufacturer protocol

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

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