SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Once in solution, store 1M DTT at -20°C.	-For optimal ChIP results, use approximately 4 X 106 cells for each immunoprecipitation to be performed. -For optimal ChIP results, it is highly critical that the chromatin is of appropriate size and concentration. -For optimal ChIP results, use approximately 5 to 10 µg of digested, cross-linked chromatin per immunoprecipitation.

Upstream tips
-Once in solution, store 1M DTT at -20°C.
Protocol tips
-For optimal ChIP results, use approximately 4 X 106 cells for each immunoprecipitation to be performed. -For optimal ChIP results, it is highly critical that the chromatin is of appropriate size and concentration. -For optimal ChIP results, use approximately 5 to 10 µg of digested, cross-linked chromatin per immunoprecipitation.

Publication protocol

According to the manufacturer’s instructions, simple ChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, Danvers, MA, USA) was applied to ChIP assays. Briefly, cells were crosslinked with EBM-2 containing 1% formaldehyde and collected in lysis buffer. And then micrococcal nuclease was used to digest the chromatids. Immunoprecipitation was incubated with 3 mg of anti-Hoxa13 antibody (1:1000, ABcam, UK) or normal rabbit IgG followed by immunoprecipitating with Protein G Agarose Beads and stored at 4 ℃ overnight with gentle shaking. Then the DNA crosslink was reversed by 5 mol/l NaCl and Proteinase K. Finally, DNA was purified. Immunoprecipitated DNAs were amplified by PCR according to their specific primers as follows:
Control PCR1: forward 5′-TGAAAATGTGGACTAGAGCCAGA-3′;
reverse 5′-CAGACACTCCAGAACAGGGC-3′;
AGGF1PCR2: forward 5′-TCCCCGCATCCATCCTCTTA-3′; reverse 5′-CCCATTCCCACTTCCTCCAC-3′.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003 below.

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