一种用于纯化人及豚鼠补体第三成分的快速快速蛋白质液相色谱法。

A rapid FPLC method for purification of the third component of human and guinea pig complement.

作者信息

Basta M, Hammer C H

机构信息

Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.

出版信息

J Immunol Methods. 1991 Aug 28;142(1):39-44. doi: 10.1016/0022-1759(91)90290-v.

DOI:10.1016/0022-1759(91)90290-v

PMID:1919020

Abstract

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.

摘要

本文描述了一种从小量血清中纯化人及豚鼠C3的方法。该程序仅需两步——聚乙二醇（PEG）沉淀和快速蛋白质液相色谱（FPLC）Mono Q HR 10/10离子交换色谱。该方案完成时间不到两小时，可得到4 - 6毫克纯化的C3。人及豚鼠成分在抗原性和功能恢复方面获得了相似的结果。从洗脱级分中回收了约67%的C3抗原，且其比活性完全保留。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和放射自显影估计，分离得到的C3纯度超过95%；使用高效价抗全人血清进行免疫电泳评估，未观察到任何明显污染，证实了这种纯度水平。该方法允许每天快速且可重复地纯化出完全活性的人或豚鼠C3。