We have a recombinant secreted glycoprotein produced in a mammalian culture system; the native protein has 12 cysteines which form 6 intramolecular disulfide bonds. We have introduced a new cysteine residue at a surface position, with the intention of targeting this residue for an in vitro site-directed chemical modification. The mutant protein is well-expressed and soluble, but while we do see some monomer, non-reducing SDS-PAGE shows that a substantial proportion of it is probably in a homodimeric form (we suspect dimerization through intermolecular disulfide formation),
Did you inhibit S-S bond formation after addition of SDS? Lots of proteins form dimers when unfolded without presence of reducing agent. Adding 20 mM NEM into SDS-PAGE loading buffer is easiest way to prevent this.
Assuming that you did this and you actually see intermolecular bond, you can play with reducing agent(s) concentrations. Intramolecular bonds require much higher concentration of reducing agents (e.g., IgG is perfectly happy and not reduced at 1 mM DTT at room temperature).
Dima
