Murine C4-binding protein: a rapid purification method by affinity chromatography.
A simple, 3-step method was described for purification of murine C4 binding protein (C4-bp), a recently recognized serum protein that functions as one of the regulatory proteins of the complement system. The method consists of 1) affinity chromatography using TNBS-BGG-conjugated Sepharose beads, 2) gel filtration on a Sepharose 6B column, and 3) heparin-Sepharose chromatography. By this method, milligram quantities of C4-bp can be easily purified by more than 500-fold from EDTA-serum of various mouse strains, and the whole purification process can be completed within 1 wk. The overall yield of C4-bp is about 15%. The C4-bp thus prepared is homogeneous as judged by SDS-polyacrylamide gel electrophoresis and immunelectrophoresis. The purified mouse C4-bp showed physicochemical properties very similar to those described for human C4-bp. Like human C4-bp, mouse C4-bp is composed of several apparently identical subunits of the m.w. of 80,000. However unlike the human counterpart, the subunits of mouse C4-bp are not linked by disulfide bonds but are connected by non-covalent forces that can be disrupted by SDS. The purified mouse C4-bp retained binding affinity for C4 and showed unaltered antigenicity. Immunization of rabbits with the purified mouse C4-bp resulted in the production of potent and monospecific antisera.
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003181:Complement C4 / Q000302:isolation & purification; D006493:Heparin / Q000494:pharmacology; D007106:Immune Sera / Q000494:pharmacology; D008297:Male; D051379:Mice; D008806:Mice, Inbred AKR; D008809:Mice, Inbred C3H; D011485:Protein Binding; D011817:Rabbits; D012685:Sepharose / Q000494:pharmacology; D013997:Time Factors; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology; D005719:gamma-Globulins / Q000494:pharmacology
D003181:Complement C4; D007106:Immune Sera; D005719:gamma-Globulins; D014302:Trinitrobenzenesulfonic Acid; D006493:Heparin; D012685:Sepharose