To examine the binding of IRF1, GCN5, and SETD2 to the SMARCC1 promoter and its upstream region, the EZ-ChIP kit (Millipore, USA) was utilized. Initially, the cells were treated with 1 % formaldehyde for 10 min to crosslink the proteins and DNA. Following this, the cells were lysed using SDS lysis buffer, and DNA fragmentation was accomplished through ultrasonication. The resulting lysates were subjected to overnight immunoprecipitation at 4 °C using specific antibodies, including IgG (1:300, ab99757, Abcam, UK), H3K4me3 (#MA5-11199, Thermo Fisher Scientific), H3K27ac (#MA5-23516, Thermo Fisher Scientific), IRF1 (ab232861, Abcam), SETD2 (#PA5-102710, Thermo Fisher Scientific), and GCN5 (#PA5-86489, Thermo Fisher Scientific). After the immunoprecipitation, the antibody–chromatin complexes were precipitated using protein G agarose at 4 °C for 1 h. Subsequently, the complexes underwent a series of washes, and the DNA was eluted. The protein-DNA complexes were then de-crosslinked, and the DNA was collected and purified for subsequent analysis using quantitative PCR (qPCR).
Ab99757
Manufactured by Abcam
Sourced in United Kingdom
Ab99757 is a laboratory equipment product. It functions as a core component for scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using ab99757
1
Investigating SMARCC1 Promoter Binding
2
DNMT3B Binding and miR-34a Interaction
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The binding relationship between DNMT3B and miR-34a was analyzed using an EZ-ChIP kit (#17-371, Millipore Corp, Billerica, MA, USA). The cells (1 × 105) were cross-linked in 1% formaldehyde for 10 min and lysed in sodium dodecyl sulfate (SDS) lysis buffer containing 40 mM Tris-Acetate, 1 mM ethylenediaminetetraacetic acid, 20 mM sodium acetate, 1% SDS, followed by sonication to shear DNA. Lysates diluted with ChIP dilution buffer were immunoprecipitated overnight at 4°C with antibodies to IgG (1:300, ab99757, Abcam, Cambridge, UK) and DNMT3B (1:100, sc-81252. Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Antibody-chromatin complexes were precipitated with 60 µL protein G agarose at 4°C for 1 h, then washed and eluted. The DNA was de-crosslinked, purified, and analyzed by real-time PCR as above mentioned.
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