SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005

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

ChIP assays were performed using the SimpleChIP enzymatic chromatin immunoprecipitation kit (Cell Signaling Technology) according to the manufacturer’s protocol with minor modification. Briefly, Meg-01 cells or rat megakaryocytes were cross-linked with 1.5% (v/v) formaldehyde for 10 minutes and then stopped with 0.125 mol/L glycine. Subsequently, nuclei were collected and chromatin sonicated to desired chromatin length (≈500 bp). Sonicated lysates were then diluted to 2 mL, with protease inhibitor cocktail, and 20 μL of this solution was removed for PCR analysis (input). After preclearing with Protein A/G Plus Agarose (Santa Cruz Biotechnology) for 1 hour at 4°C, antibody specific to nuclear factor (NF)-κB p65 subunit was added and incubated at 4°C overnight on a rocking platform. Immune complexes were collected by the addition of Protein A/G Plus Agarose for 1 hour at 4°C, and the agarose beads were extensively washed with solutions of increasing ionic strength. Bound immune complexes were eluted and cross-links were reversed by incubating at 65°C for 4 hours. Samples were then treated with proteinase K, and DNA was purified using DNA purification spin columns.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005 below.

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