Isolation of human complement subcomponents C1r and C1s in their unactivated, proenzyme forms.
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.
Lane
P D
PD
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Schumaker
V N
VN
Tseng
Y
Y
Poon
P H
PH
D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D015923:Complement C1r / Q000302:isolation & purification*; D003173:Complement C1s / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000302:isolation & purification*; D006801:Humans
D004792:Enzyme Precursors; D015923:Complement C1r; D003173:Complement C1s