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

To explore the regulatory mechanism of stathmin in OSCC, ChIP was conducted in OSCC cell lines. ChIP assays were performed according to the manufacturer’s instructions using the SimpleChIP Enzymatic Chromatin IP Kit (magnetic beads; CST). Briefly, p53 and protein complexes were cross-linked inside HN6, HN13 and HN30 cells by the addition of formaldehyde (1% final concentration) to the cells in culture. Chromatin was digested with micrococcal nuclease and sonicated into 150–900 bp DNA/protein fragments. An aliquot of the cross-linked protein complexes was immunoprecipitated by incubation with either p53-specific antibody (CST) or IgG antibody overnight at 4 °C with rotation. Chromatin-antibody complexes were isolated from solution by incubation with ChIP-Grade Protein G Magnetic Beads for 1 h at 4 °C with rotation. The bead-bound immune complexes were then washed and eluted from the beads with elution buffer. Eluates were heated at 65 °C overnight to reverse the formaldehyde cross-linking and DNA was extracted. DNA samples from chromatin immunoprecipitation preparations were analyzed by PCR using primers spanning the stathmin gene (NM_203401.1) in the promoter region (forward, 5′-AATGGGGAGCTGGTTCGGA-3′; reverse, 5′-GTGTAGTCCTGTCCCGGAGG-3′). The transcription factor binding sites of p53 were predicted using the website of JASPAR (http://jaspar.genereg.net/) [27] and PROMO [28, 29]. The two predicted binding sites of CCAGGCCCACACCTG and GCAACCCCCGGCATT were amplified using the above promoter primers. Primers used in the real-time PCR mentioned above amplified the CDS region. The nonpromoter regions were used as control.

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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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