Van den Berg C W, Van Dijk H, Capel P J
Section of Experimental Immunology, Medical Faculty, State University, Utrecht, The Netherlands.
J Immunol Methods. 1989 Aug 15;122(1):73-8. doi: 10.1016/0022-1759(89)90336-0.
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.
本文描述了一种快速的、为期1天的小鼠补体因子C3和C5的纯化方法。该方法基于聚乙二醇6000分级沉淀，随后在快速蛋白质液相色谱（FPLC）系统上进行Mono Q阴离子交换色谱。对于C3的分离，还使用了Superose 12（凝胶过滤）进行额外的FPLC分离步骤。通过生物活性测定，C3纯化了71倍，产率为32%；通过SDS-PAGE判断，该制剂不含可检测到的污染物。分离C5的类似方法得到的制剂有相当程度的污染，使用针对这些污染物的抗体进行亲和色谱可以很容易地去除。通过这种联合方法，基于生物活性，C5纯化了536倍，产率为28%。SDS-聚丙烯酰胺凝胶电泳显示，小鼠C3和C5的表观分子量分别为170,000和190,000。在还原条件下，C3的α链和β链分子量分别为107,000和62,000，C5的α链和β链分子量分别为104,000和85,000。
