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 chemokine with four cysteines, aggregated during the stacking phase of SDS-PAGE and generated a band with an apparent molecular mass of 18 kDa. This aggregation 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 could be prevented by reduction of its sulfhydryls with dithiothreitol followed 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 purity.
University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
M K
MK
Crow
N
N
Karasavvas
A H
AH
Sarris
D054357:Chemokine CXCL10; D019743:Chemokines, CXC / Q000737:chemistry*; D003545:Cysteine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D010084:Oxidation-Reduction
D054357:Chemokine CXCL10; D019743:Chemokines, CXC; D003545:Cysteine