Re-ChIP Protocol
- 4 steps
- 1 hours
- 3 reagents required
Re-ChIP stands for sequential chromatin immunoprecipitations with two antibodies to study the simultaneous presence of two proteins, or different histone modifications in the genome sequence of interest. We perform elution with Elution Buffer (contains 1% SDS). We found it crucial to immobilize antibodies on protein A/G beads to avoid leakage of antibodies from the first ChIP to the second ChIP. We incubate a fraction of eluate from the first ChIP with empty beads as an antibody leakage control.
Reagents Required
| Product | Preparation |
| DTT | Prepare 10 mM solution in molecular biology-grade water. |
| Sonication Buffer | 50 mM Hepes, pH 7.9 140 mM NaCl 1 mM EDTA 1% Triton X-100 0.1% Na-deoxycholate 0.1% SDS 0.5 mM PMSF Protease inhibitor cocktail (Roche) |
| Elution Buffer | 50 mM Tris, pH 8.0 1 mM EDTA 1% SDS 50 mM NaHCO3 |
Procedure
-
After step 24 of the ChIP protocol, incubate the beads with an equal volume of 10 mM DTT for 30 minutes at 37°C.
-
Centrifuge at 14,000 rpm for 1 minute and transfer the supernatant into a new tube.
-
Repeat step 1 and combine the eluates.
-
Dilute the eluted sample 40 times with sonication buffer. Keep 10% of the sample for input and proceed with step 18 of the ChIP protocol by adding the second antibody.