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 was performed in kidneys from wild-type SV129 mice injected with NTS or PBS (control), using the SimpleChIP Plus Enzymatic Chromatin IP Kit (#9005; Cell Signaling Technology) per manufacturer’s instructions. Briefly, whole kidneys were finely minced and crosslinked with 1.5% formaldehyde for 20 minutes under constant rotation, followed by addition of 0.1 M glycine to stop the reaction. The tissue was disaggregated using a Dounce homogenizer, nuclei were prepared, and chromatin was digested, sonicated, and analyzed by gel electrophoresis and OD260 measurement to determine DNA concentration. Precipitation was performed with the following ChIP-graded antibodies from Cell Signaling Technology: NF-κB p65 (#8242), p-c-Jun (#3270), p-Stat1 (#7649), and p-Smad3 (#9520). Rabbit IgG was used as control. The precipitated complexes were purified with the use of Protein G Magnetic Beads, crosslinks were reversed, and DNA was eluted and purified. Sequence-specific primers for selected regions of mouse periostin promoter (Supplemental Table 3) were used in subsequent quantitative real-time PCR analysis to check for enrichment of precipitated DNAs in the NTS over the PBS group. The results were expressed as percentage of the input (total DNA used for immunoprecipitation).

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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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