Lane P D, Schumaker V N, Tseng Y, Poon P H
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
J Immunol Methods. 1991 Aug 9;141(2):219-26. doi: 10.1016/0022-1759(91)90148-9.
We have modified a standard isolation procedure for C1r and C1s, which employs IgG-Sepharose affinity chromatography followed by DEAE chromatography. As usual, all steps were performed at low temperature and two proteolytic inhibitors, PMSF and NPGB, were added during affinity chromatography on IgG-Sepharose. The novel condition was to keep the pH at pH 6.1 during the entire procedure, where activation was markedly depressed. In addition, purification was improved by washing the IgG-Sepharose column with a buffer free of added divalent cations immediately prior to elution of the C1r and C1s with EDTA. The final yields of highly purified C1r and C1s were about 20%; little or no activated material was detected in these highly purified fractions.
我们改进了一种用于分离C1r和C1s的标准方法，该方法采用IgG-琼脂糖亲和层析，随后进行DEAE层析。与往常一样，所有步骤均在低温下进行，并且在IgG-琼脂糖亲和层析过程中添加了两种蛋白水解抑制剂PMSF和NPGB。新的条件是在整个过程中将pH保持在6.1，在此pH下活化作用明显受到抑制。此外，在用EDTA洗脱C1r和C1s之前，立即用不含添加二价阳离子的缓冲液洗涤IgG-琼脂糖柱，从而提高了纯化效果。高纯度C1r和C1s的最终产率约为20%；在这些高纯度级分中几乎检测不到或未检测到活化物质。
