Rapid isolation and characterization of native mouse complement components C3 and C5.
A rapid, 1 day procedure for the purification of mouse complement factors C3 and C5 is described. The method is based on fractionated precipitation by polyethylene glycol 6000, followed by Mono Q anion exchange chromatography on a system for fast protein liquid chromatography (FPLC). For C3 isolation, an additional FPLC separation step using Superose 12 (gel filtration) was used. C3 was purified 71-fold with a yield of 32% as measured by biological activity; the preparation contained no detectable contaminants as judged by SDS-PAGE. A comparable procedure for the isolation of C5 resulted in a preparation with a considerable contamination which could be easily removed by affinity chromatography using antibodies directed against these contaminants. With this combined procedure C5 was purified 536-fold with a yield of 28% based on biological activity. SDS-polyacrylamide gel electrophoresis revealed that mouse C3 and C5 had apparent Mrs of 170,000 and 190,000, respectively. Under reducing conditions the alpha and beta chains showed Mrs of 107,000 and 62,000 for C3, and 104,000 and 85,000 for C5.
Section of Experimental Immunology, Medical Faculty, State University, Utrecht, The Netherlands.
C W
CW
Van den Berg
H
H
Van Dijk
P J
PJ
Capel
D000818:Animals; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D003176:Complement C3 / Q000302:isolation & purification* / Q000502:physiology; D003182:Complement C5 / Q000302:isolation & purification* / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D013997:Time Factors
D003176:Complement C3; D003182:Complement C5