Visualization of lipoprotein receptors by ligand blotting.
Visualization of lipoprotein receptors by ligand blotting.
复制标题
DOI：
10.1016/s0021-9258(18)32666-8
复制
发表时间：
1983-04
期刊：
影响因子：
--
通讯作者：
T O Daniel;W J Schneider;J. L. Goldstein;M. Brown
中科院分区：
文献类型：
--
作者：
T O Daniel;W J Schneider;J. L. Goldstein;M. Brown
本文描述了通过配体印迹对低密度脂蛋白 (LDL) 受体进行可视化。将去污剂溶解的膜制剂进行一维或二维十二烷基硫酸钠 (SDS)-聚丙烯酰胺凝胶电泳，然后将蛋白质转移到硝酸纤维素纸上。将纸与天然 LDL 一起孵育，然后与针对 LDL 的 125 I 标记抗体一起孵育，并通过放射自显影观察结合的抗体。 LDL 印迹的成功取决于电泳系统中巯基还原剂的省略。即使在 SDS 存在的情况下进行电泳后，链内二硫键仍使受体保持其结合活性。在鉴定 LDL 受体时，配体印迹技术与针对 LDL 受体的单克隆抗体的免疫印迹技术一样灵敏。因此，当没有抗受体抗体时，它可以用于识别受体。我们利用该技术证明兔肾上腺的LDL受体与牛肾上腺皮质和人成纤维细胞的LDL受体具有相同的分子量。配体印迹技术通常适用于 SDS 凝胶电泳后其他质膜受体的可视化。
This paper describes the visualization of the low density lipoprotein (LDL) receptor by ligand blotting. Preparations of detergent-solubilized membranes are subjected to one- or two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, after which the proteins are transferred to nitrocellulose paper. The paper is incubated with native LDL and then with an 125I-labeled antibody against LDL, and the bound antibody is visualized by autoradiography. The success of LDL blotting depends on the omission of sulfhydryl reducing agents from the electrophoresis system. Intrachain disulfide bonds allow the receptor to retain its binding activity even after electrophoresis in the presence of SDS. In identifying LDL receptors, the ligand blotting technique is as sensitive as immunoblotting with a monoclonal antibody against the LDL receptor; it can therefore be used to identify receptors when no anti-receptor antibodies are available. We use this technique to show that the LDL receptor of the rabbit adrenal gland has the same molecular weight as the LDL receptor of the bovine adrenal cortex and human fibroblasts. The ligand blotting technique may be generally applicable for visualization of other plasma membrane receptors after SDS-gel electrophoresis.