Lin P H, Selinfreund R H, Wakshull E, Wharton W
University of California Experimental Pathology Group, Los Alamos National Laboratory, New Mexico 87545.
Anal Biochem. 1988 Feb 1;168(2):300-5. doi: 10.1016/0003-2697(88)90322-3.
A rapid method for the purification of plasma membrane from a relatively small number of A431 cells is described. The method is a simple, two-step differential centrifugation in the presence of Ca2+ that requires a total centrifugation time of 7 min. The membrane preparations contained a high level of epidermal growth factor (EGF) receptor activity demonstrated by both the quantity of specific ligand binding and the amount of EGF-dependent phosphorylation of the receptor and an exogenous substrate. EGF-dependent autophosphorylation identified the EGF receptor in the purified membranes as an undegraded 170-kDa protein.
本文描述了一种从相对少量的A431细胞中快速纯化质膜的方法。该方法是在Ca2+存在下进行的简单两步差速离心，总离心时间为7分钟。通过特异性配体结合量以及受体和外源底物的EGF依赖性磷酸化量证明，膜制剂含有高水平的表皮生长因子（EGF）受体活性。EGF依赖性自磷酸化将纯化膜中的EGF受体鉴定为未降解的170 kDa蛋白。
