C5a generation by thrombin and C5 convertase. Reactions containing C5 (400nM) were incubated with thrombin (IIa) (20nM or 400nM) or C5 convertase (CVF,Bb; 100nM) alone or in combination in HBS-M₇, pH 7.4, for 15 minutes, 60 minutes, or 20 hours at 37°C. Reactions were subjected to 15% Tris-tricine SDS-PAGE under reducing conditions. C5a was detected by Western blotting using a polyclonal rabbit antibody against C5a. Results are representative of 3 independent experiments, each with similar results. Inset: C5a in-gel standards from Western blot and corresponding standard curve.