ChIP (Chromatin immunoprecipitation) assay
This protocol is extracted from research article:
HES1 promotes breast cancer stem cells by elevating Slug in triple-negative breast cancer
Int J Biol Sci, Jan 1, 2021; DOI: 10.7150/ijbs.53477

Potential transcription factor HES1-binding sequences within Slug promoter region were identified by JASPAR database. According to the protocol of ChIP assay kit (Merk Millipore); MDA-MB-231 cells were cross-linked with 1% formaldehyde at 37 °C for 10 min after washing. Cells were resuspended in lysis buffer, sonicated to shear DNA and sonicates were immunoprecipitated with anti-HES1 antibody (ab70576, Abcam) or negative control IgG at 4 °C overnight. The immunocomplex was collected on protein A/G-agarose and washed in turn with low salt, high salt, lithium chloride buffer and TE buffer. After elution and reverse cross-linking, the DNA was extracted and analyzed by PCR using primer pairs for the Slug promoter: Slug (BE1) forward: 5′-CAAGGAGGACTCCTGCTCTC-3′, reverse: 5′-CCTCTGGCTTTTACTCCAGG-3′; Slug (BE2) forward: 5′-GCTAACACGGTGACATGAGT-3′, reverse: 5′-CACGCAAGGCTGCAGT-3′; Slug (BE3) forward: 5′- CCCTCCTAGCTCCCAGA-3′, reverse: 5′-CCTCTCCACTGAAATCTCAA-3′.

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