A rapid FPLC method for purification of the third component of human and guinea pig complement.
A method is described for the purification of human and guinea pig C3 from small amounts of serum. This procedure requires only two steps--polyethylene glycol (PEG) precipitation and fast protein liquid chromatography (FPLC) Mono Q HR 10/10 ion exchange chromatography. The protocol takes less than two hours to complete and yields 4-6 mg of purified C3. Similar results, in terms of antigenic and functional recovery, were obtained for both human and guinea pig components. About 67% of C3 antigen was recovered from eluted fractions with fully preserved specific activity. Isolated C3 was over 95% pure as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography; this level of purity was confirmed by the absence of any observable contamination as assessed by immunoelectrophoresis using high titer anti-whole human serum. This method allows rapid and reproducible purification of fully active human or guinea pig C3 on a daily basis.
Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
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D000818:Animals; D001345:Autoradiography; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003176:Complement C3 / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006801:Humans; D007122:Immunoelectrophoresis; D015203:Reproducibility of Results
D003176:Complement C3