Hi Min, Given the limited supply of your protein you could try the EDC-NHS zero-length crosslinking either in one-step or two-step protocol (Anal. Biochem. 185,131,1990). If your protein complex is like most protein complexes chances are there will be some electrostatic contacts at the interface (Glu-Lys or Asp-Lys). These can be crosslinked and you need only micrograms of your protein to test it. The caveat here is that crosslinked proteins usually do not migrate on SDS-PAGE according to their Mr., but you can get a pretty good idea from the pattern of crosslinking at various protein ratios and by changing the extent of crosslinking. You can also label one of your proteins with a fluorescent probe (SH or NH2 specific) and determine stoichiometry form the change in fluorescence/Coomassie staining of the crosslinked products as compared to the labeled monomer.
Good luck Zenon
