ChIP Assay Protocol with Modifications
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Corresponding Organization : University of Pittsburgh
Other organizations : Johns Hopkins Medicine, Johns Hopkins University
Variable analysis
- Zymo-Spin™ ChIP kit used with modifications based on published protocols
- Atlantis MNase (Zymo Research) used to digest chromatin
- ChIP grade mouse monoclonal antibody against human TBX21 (4B10 and D39) (Santa Cruz Biotech) used
- Control mouse IgG antibody (Santa Cruz Biotech) used
- Promoter region of the target gene identified by PCR with sets of primers
- Cell number (5–6 ×10^6 cells) used
- Fixation with 1% paraformaldehyde for 10min at 37°C
- Stopping fixation with 2.5M glycine
- Nuclei extraction
- Mild sonication to release digested chromatin
- Incubation of digested chromatin with antibodies overnight at 4°C
- Immune-precipitation with MagnaChIP protein A+G magnetic beads (EMD Millipore)
- Washes after immune-precipitation
- Storage of bound DNA at −80°C until PCR
- None explicitly mentioned
- Control mouse IgG antibody (Santa Cruz Biotech)
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