>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
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