Figure 9
Digestion of CnPC with Protac. (A) Silver stained non-reduced 12% SDS-PAGE depicting the plasma protein fractions obtained over the course of purification of protein C from canine plasma. Five ÎĽg of protein C sample (zymogen or activated) was loaded into each lane. Lane 1: Molecular weight markers (3 ÎĽL). Lane 2: CnPC purified from canine plasma. Lane 3: CnPC following incubation with Protac, a specific activator of protein C; note the decrease in MW of activated CnPC (Ac) and small amount of undigested zymogen form (Zm). Lane 4: Protac purchased commercially. (B) Western blot of the adjacent SDS-PAGE (A) with a polyclonal antibody against the peptide EDQVDPRLIDGK found in human protein C zymogen, but not in the activated form of protein C. Lane loads were the same as (A). An immunoreactive protein fraction was detected in the undigested sample. Following incubation with Protac, the amount of immunoreactive protein (Zm) was significantly decreased. The activated form of the protein (Ac) was, as predicted, not immunoreactive to the primary antibody.