Protein aggregation mediated by cysteine oxidation during the stacking phase of discontinuous buffer SDS-PAGE

The resolution of complex protein mixtures by discontinuous buffer SDS-PAGE is accomplished by their concentration into thin bands in the stacking gel , followed by their separation during migration through the resolving gel. Recombinant human interferon-inducible protein-10 (IP-10), a 10-kDa C-X-C c hemokine with four cysteines, aggregated during the stacking phase of SDS-P AGE and generated a band with an apparent molecular mass of 18 kDa. This ag gregation depended on the presence of reduced sulfhydryl residues on IP-10, on the amount of loaded protein, and on the concentration of the ammonium persulfate used to polymerize the stacking gel. The aggregation of IP-10 co uld be prevented by reduction of its sulfhydryls with dithiothreitol follow ed by irreversible blockade with iodoacetamide. These methods may be useful in the prevention of aggregation of sulfhydryl-containing proteins during SDS-PAGE, especially when large quantities are analyzed to assess their pur ity.