Purification of the third component of canine complement.
A rapid, simple procedure has been developed for the purification of the third component (C3) of canine complement. Dog plasma was initially fractionated by precipitation with 4% (w/v) polyethylene glycol (PEG) 4000. The supernatant was depleted of plasminogen using a Sepharose 4B-lysine column, and the effluent was again fractionated with PEG 4000 at 16% (w/v). The precipitate was resuspended and passed over a DEAE-Sephacel column. The fractions containing hemolytically active C3 were pooled, concentrated, and passed over a CM-Sepharose CL-6B column to yield the final purified product. Rabbit anti-whole dog serum identified only one protein in the purified material on immunoelectrophoresis. When injected into a rabbit, the purified product raised an antisera which reacted with only one protein in both whole dog serum and the purified product. Analysis by SDS-PAGE revealed a single band of MW 179,000 +/- 7,000 (+/- 1 S.D.) daltons which, upon reduction with 2-mercaptoethanol, resulted in 2 bands of 114,000 +/- 6,000 daltons and 65,000 +/- 3,500 daltons. Final recovery was 18% with respect to C3 antigen and 9% with respect to C3 hemolytic activity.
D000818:Animals; D002845:Chromatography / Q000379:methods; D003176:Complement C3 / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D004285:Dogs / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D005590:Fractional Precipitation; D006461:Hemolysis; D007122:Immunoelectrophoresis
D003176:Complement C3