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)


  1. Wash cells 3 times with ice-cold PBS.
  2. 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.
  3. Incubate on ice for 30 minutes.
  4. Wash cells twice with cold PBS.
  5. Add 20–25 mL ice-cold quenching buffer per plate.
  6. Incubate on ice for 10 minutes.
  7. Take the plates out from ice and wash 3 times with PBS at room temperature.
  8. Add 27 mL PBS to each plate + 3 mL 10% formaldehyde. Mix well and incubate at room temperature for 10 minutes.
  9. Add 3 mL glycine. Mix well.
  10. Wash 3 times with cold PBS/0.5 mM PMSF and continue with ChIP protocol.