Bifunctional Cross-Linking Protocol
- 10 steps
- 2 hours
- 6 reagents required
A general observation in ChIP assays is that the signals obtained for histones and DNA-binding factors are much stronger than those proteins that do not directly contact DNA but are recruited via protein-protein interactions. There could be several explanations for this phenomenon. One of them is cross-linking efficiency by formaldehyde. In our experience, ChIP signals for chromatin remodeling complexes can be improved 2–4 times by using dimethyl-3,3'-dithiobispropionimidat (Thermo Scientific Pierce-DTBP ) in conjunction with formaldehyde. The protocol was adopted from Fujita & Wade, 2004 and is described for cells grown in 150 mm dish.
Reagents Required
| Product | Preparation |
| PBS (Phosphate Buffered Saline) | Use 10X PBS, pH 7.2 (0.2 M Potassium Phosphate, 1.5 M NaCl). Dilute appropriate volume to 1X with molecular biology-grade water. |
| DTBP (Dimethyldithiobispropionimidat) | |
| Quenching Buffer | Prepare using 100 mM Tris pH 8.0, 150 mM NaCl. |
| 10% Formaldehyde | |
| Glycine (1.375 M) | |
| PMSF (Phenylmethansulfonylfluorid) |
Procedure
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Wash cells 3 times with ice-cold PBS.
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Prepare (freshly) 5 mM DTBP in ice-cold PBS and add to plates sitting on ice.
Note: To cover cells, 20–25 ml of this solution is needed per 150 mm plate. -
Incubate on ice for 30 minutes.
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Wash cells twice with cold PBS.
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Add 20–25 mL ice-cold quenching buffer per plate.
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Incubate on ice for 10 minutes.
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Take the plates out from ice and wash 3 times with PBS at room temperature.
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Add 27 mL PBS to each plate + 3 mL 10% formaldehyde. Mix well and incubate at room temperature for 10 minutes.
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Add 3 mL glycine. Mix well.
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Wash 3 times with cold PBS/0.5 mM PMSF and continue with ChIP protocol.