Chromatin Immunoprecipitation (ChIP) Assay Kit

Protocol tips

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	107 cells were cultivated in DMEM with 10% FBS in the absence or presence of 10 nM leptin for 1 hour. Cells were washed in 1X PBS and incubated with 1% formaldehyde for 10 minutes. Cells were lysed and sonicated using a Branson digital sonifier with 30% impulse power for six impulses. For immunoprecipitation, precleared cell lysates were incubated with anti–P-Y705-STAT3 (9131; Cell Signaling Technology) or mouse IgG (Santa Cruz Biotechnology Inc.) as a negative control.

Protocol tips

107 cells were cultivated in DMEM with 10% FBS in the absence or presence of 10 nM leptin for 1 hour. Cells were washed in 1X PBS and incubated with 1% formaldehyde for 10 minutes. Cells were lysed and sonicated using a Branson digital sonifier with 30% impulse power for six impulses. For immunoprecipitation, precleared cell lysates were incubated with anti–P-Y705-STAT3 (9131; Cell Signaling Technology) or mouse IgG (Santa Cruz Biotechnology Inc.) as a negative control.

Publication protocol

ChIP was performed according to the manufacturer’s guidelines (17-295; Millipore). Briefly, 107 cells were cultivated in DMEM with 10% FBS in the absence or presence of 10 nM leptin for 1 hour. Cells were washed in 1X PBS and incubated with 1% formaldehyde for 10 minutes. Cells were lysed and sonicated using a Branson digital sonifier with 30% impulse power for six impulses. For immunoprecipitation, precleared cell lysates were incubated with anti–P-Y705-STAT3 (9131; Cell Signaling Technology) or mouse IgG (Santa Cruz Biotechnology Inc.) as a negative control. Lysates were washed, eluted in Tris-EDTA buffer, and reverse cross-linked using 1% SDS and 0.1 M NaHCO3. For PCR amplification of the target sequences, the following primers were used: P1: 5′-AGAAAATGCCGCGCTCCCTAC-3′; P2: 5′-GAAAGACTCGGAGGCGGAAGAA-3′; negative control A: 5′-CACTTGGAGGATACCAGAGCA-3′; negative control B: 5′-GCTCTCCAGGGTTACCTGAGC-3′.

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Manufacturer protocol

Download the product protocol from Merck Millipore for Chromatin Immunoprecipitation (ChIP) Assay Kit below.

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