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

Cells were fixed with fresh 1% formaldehyde (final concentration) in Medium199 for 10 min at room temperature. Fragmented chromatin was prepared using SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling Technologies no. 9003). Chromatin immunoprecipitation was performed with rabbit anti-p-STAT1 (Tyr701; Cell Signaling Technologies no. 9167, 1:80), rabbit anti-histone H3 (a technical positive control Cell Signaling Technologies no. 4620, 1:40) and normal rabbit IgG (a negative control, Cell Signaling Technologies no. 2729, 2.5 μg/ml). After reverse cross-linking and DNA purification, immunoprecipitated DNA was quantified by real-time PCR using SYBR Green (Roche no. 04673484001) with primers for a STAT1 binding site in the JNK3 promoter (forward primer 5′- GGTTTCCAGGCAGTGAAAGA-3′, reverse primer 5′- GAATTGAGGGGTGAGGACAA-3′), interferon regulatory factor 1 (forward primer 5′- AAGAGGGAAGAAGGCAGAGG-3′, reverse primer 5′- GGGAATCCCGCTAAGTGTTT-3′) and RPL30 exon 3 (Cell Signaling Technologies no. 7014). Fold enrichment was calculated based on Ct value of IgG control using the comparative Ct method.

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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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