Steffen W, Hodgkinson J L, Wiche G
University of Vienna, Institute of Biochemistry and Molecular Cell Biology, Biocenter, Dr. Bohrgasse 9, A-1030, Vienna, Austria.
J Struct Biol. 1996 Nov-Dec;117(3):227-35. doi: 10.1006/jsbi.1996.0087.
It was our goal to determine the location of the intermediate chain within the complex of cytoplasmic dynein by immunoelectron microscopy. To do so we generated two monoclonal antibodies (m74-1 and m74-2) specific for the intermediate chain. Both antibodies recognised the intermediate chain by sodium dodecyl sulphate-polyacrylamide gel electrophoresis immunoblot and ELISA assays of native and denatured proteins. When sucrose density gradient-purified cytoplasmic dynein from bovine brain was incubated with the gold-conjugated monoclonal antibodies, m74-1 and m74-2, and examined by negative staining, the gold label was found opposite the globular heads at the base of the V-shaped stalk of the motor complex. The labelling of the intermediate chain is the first mapping of a component within cytoplasmic dynein. The identification of the intermediate chain at the base of the complex supports a possible docking function of the intermediate chain.
我们的目标是通过免疫电子显微镜确定中间链在胞质动力蛋白复合体中的位置。为此，我们制备了两种针对中间链的单克隆抗体（m74 - 1和m74 - 2）。两种抗体通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳免疫印迹以及对天然和变性蛋白质的酶联免疫吸附测定法识别中间链。当将来自牛脑的经蔗糖密度梯度纯化的胞质动力蛋白与金标记的单克隆抗体m74 - 1和m74 - 2一起孵育，并通过负染色进行检查时，发现金标记位于运动复合体V形柄基部的球状头部相对的位置。中间链的标记是胞质动力蛋白内一个组分的首次定位。在复合体基部鉴定出中间链支持了中间链可能具有对接功能的观点。
