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

Analysis of TCF7L2 and HNF4α genome-wide occupancy was carried out using specific and internally-validated antibodies. For TCF7L2, we used an antibody (clone C48H11, cat. #2569, Cell Signaling Technology, Danvers, MA, USA), that has been previously validated by us for use in western blotting, immunoprecipitations and ChIP-Seq (9) and, more recently, by the Encode Consortium (19). This antibody recognizes a region surrounding Leu330 of the TCF7L2 protein, which is at the 3′ end of exon 9 and is common to all known and theoretical Tcf7l2 mRNA transcripts. It should be noted, however, that only two major protein isoforms are observed in the H4IIE cells, both of which are detected approximately equally by #2569 on western blots. For HNF4α, a mouse monoclonal antibody (clone H1415, cat. #PP-H1415–00, R&D Systems, Minneapolis, MN, USA) was selected for ChIP-Seq analysis and was validated using the rat apolipoprotein-C-III (Apoc3) gene promoter. A Myc-tag antibody (clone 71D10, cat. #2278, Cell Signaling Technology) was used as a negative control (Supplementary Figure S1). ChIP-Seq was performed on scramble and siRNA-Tcf7l2 treated cells in duplicate (for TCF7L2) and each sequenced sample (for HNF4α and TCF7L2) represented a pool of two independent immunoprecipitations, each consisting of between 10 and 20 μg of chromatin.

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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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