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 assay was performed to screen for androgen receptor (AR)-binding sites in the promoter region of the ins1 gene. The assay was performed using a SimpleChIP ChIP kit (#9005; Cell Signaling Technologies) following the manufacturer's instruction. Briefly, pancreatic tissues from control and DHT-treated female rats were washed and cross linked by incubating them with 1% formaldehyde for 10 min at room temperature. Cross linking was stopped by the addition of 1X glycine for 5 min. After washing with ice-cold PBS, tissues were lysed to prepare chromatin. The chromatin was then fragmented by enzymatic digestion using micrococcal nuclease for 20 min at 37°C with frequent mixing. The digestion was stopped by adding 0.5 M EDTA on ice. Next, the nuclei were pelleted and sonicated for 20 s twice with 40% power to break the nuclear membrane. The lysates were then clarified by spinning at 10 000 rpm for 10 min at 4°C. The supernatant contained the required cross linked chromatin. The chromatin was then treated with RNase A and then incubated with Proteinase K for 2 h at 65°C. DNA from the digested chromatin was isolated using spin columns, quantified, and run on a 1% agarose gel to verify the enzymatic digestion of chromatin. Chromatin (5 μg of DNA in 500 μl volume for each immunoprecipitation) was then subjected to immunoprecipitation with an AR antibody (25 μl per reaction, Abcam; ab74272) along with positive (10 μl anti-histone antibody; Cell Signaling Technologies #4620) and negative (1 μl of rabbit IgG, Cell Signaling Technologies #2727) controls. Immunoprecipitation was performed overnight at 4°C with constant rotation. ChIP-grade Protein G magnetic beads were then added to each reaction and incubated for 2 h at 4°C with rotation. Next, the pellets were precipitated using a magnetic rack. Cross links were then reversed by adding 5 mM NaCl and proteinase K and incubating at 65°C for 2 h. DNA was purified using spin columns, quantified, and stored for further analysis. Purified DNA from ChIP assays along with their respective 2% inputs were subjected to qPCR to identify the ligand-dependent AR binding to putative AR-binding sites in ins1 gene. Primer sets were synthesized to flank the predicted putative ARE sites (Table S1). PCR conditions used were 10 min at 95°C for 1 cycle, 15 s at 95°C, 30 s at 60°C, and 15 s at 72°C for 40 cycles, with a final dissociation step (0.05 s at 65°C and 0.5 s at 95°C). Percentage Input was calculated by calculating 2% × 2(C[T] 2% Adjusted Input Sample—C[T] IP Sample) (C[T] = Threshold cycle of PCR reaction).

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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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