The detection of multimeric forms of phospholipase A2 upon sodium dodecylsulfate-polyacrylamide electrophoresis.
The detection of multimeric forms of phospholipase A2 upon sodium dodecylsulfate-polyacrylamide electrophoresis.
复制标题
通过十二烷基硫酸钠-聚丙烯酰胺电泳检测多聚体形式的磷脂酶 A2。
DOI：
--
复制
发表时间：
1991
期刊：
影响因子：
--
通讯作者：
H. Van den bosch
中科院分区：
文献类型：
--
作者：
C. Schalkwijk;F. Märki;I. Wiesenberg;H. Van den bosch
抗大鼠肝线粒体磷脂酶A2的单抗与酪蛋白诱导的大鼠腹膜渗出液中的磷脂酶A2在斑点印迹和单抗-琼脂糖结合实验中均有交叉反应。对渗出物酶进行免疫亲和纯化，得到酶制剂，估计在单层析步骤中浓缩约54000倍。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法显示，除一条14 KDa的磷脂酶A2区带外，还有几条相对分子质量较高的区带。经Western blotting和免疫染色后，只检测到一个14 kDa的磷脂酶A2，但样品在十二烷基硫酸盐和二硫代树突醇中储存后，出现了这种磷脂酶A2的相关形式。从大鼠血小板和大鼠肝线粒体获得的免疫纯化和均一的磷脂酶A2制剂也观察到类似的形成多聚体的趋势。这一现象可能对解释用抗磷脂酶A2抗体在蛋白质印迹试验中获得的结果具有普遍意义。
Monoclonal antibodies against rat liver mitochondrial phospholipase A2 were found to be cross-reactive with the phospholipase A2 present in caseinate-induced rat peritoneal exudate, both in dot-blot and in monoclonal antibody-Sepharose binding experiments. Immunoaffinity purification of the exudate enzyme yielded enzyme preparations with an estimated enrichment of about 54000-fold in a single chromatographic step. Sodium dodecylsulfate-polyacrylamide gelectrophoresis showed, apart from the presence of a 14 KDa phospholipase A2 band, several other bands at higher molecular weights. After Western blotting and immunostaining only a 14 kDa phospholipase A2 was detected, but upon storage of the sample in dodecylsulfate and dithiotreitol associated forms of this phospholipase A2 appeared. Similar tendencies to form multimers were observed starting from immunopurified and homogeneous phospholipase A2 preparations obtained from rat platelets and from rat liver mitochondria. This phenomenon is likely to be of general importance for the interpretation of results obtained in Western blots using antiphospholipase A2 antibodies.