SimpleChIP® Plus Enzymatic Chromatin IP Kit (Agarose Beads) #9004

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 with SimpleChIP Enzymatic Chromatin IP Kit (Agarose Beads, Cell Signaling Technology) according to the manufacturer's instructions. In brief,
after stimulation with LPS (200 ng/ml) for 4 hours, BMDCs were cross-linked in 1%
(w/v) formaldehyde (Sigma-Aldrich) and lysed for nuclei. Then the nuclei were digested
with micrococcal nuclease and sonicated with Ultrasonic processor (FS-150N, 30%
amplitude, 5 sets of 20 sec pulse) for 150-900 bp chromatin fragments. A portion of
sonicated nuclei was saved as input control. Other sonicated nuclei were incubated with 2
g anti–c-Jun (#9165S, Cell Signaling Technology) or negative control IgG overnight at
4oC followed with incubation with ChIP-Grade Protein G Agarose Beads for 2 hours at 4 oC. Then chromatin was eluted by incubation for 30 min at 65oC and reverse cross-link
by Proteinase K for 2 hours at 65oC. Finally, DNA was purified for gene promoter region
amplifier by qRT-PCR.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for SimpleChIP® Plus Enzymatic Chromatin IP Kit (Agarose Beads) #9004 below.

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