Matthaei S, Baly D L, Horuk R
Anal Biochem. 1986 Aug 15;157(1):123-8. doi: 10.1016/0003-2697(86)90205-8.
A method describing the rapid and effective transfer of integral membrane protein from isoelectric focusing gels to nitrocellulose is described. Initial experiments were carried out with detergent-solubilized extracts of human erythrocyte membrane proteins. The effectiveness of the transfer was demonstrated by assaying for erythrocyte glucose transporter, an integral membrane protein, using specific antibodies followed by 125I-protein A and autoradiography. Several detergents including octyl glucoside, Triton X-100 and CHAPS were used in this study but only octyl glucoside effectively solubilized the glucose transporter and did not interfere with the electrotransfer of the protein. The glucose transporter separated on isoelectric focusing gels was effectively transferred after 2 h of electroblotting and was found to have an apparent pI of 6.4-6.5. These findings were substantiated by photolabeling red cell membranes with [3H]cytochalasin B in the presence or absence of D-glucose (which inhibits [3H]cytochalasin B binding to the glucose transporter) and separating the labeled proteins by two dimensional electrophoresis. With this procedure we identified a D-glucose sensitive 50-60 kDa protein focusing with an apparent pI of around pH 6.4-6.5.
本文描述了一种将整合膜蛋白从等电聚焦凝胶快速有效地转移至硝酸纤维素膜的方法。最初的实验是用人红细胞膜蛋白的去污剂溶解提取物进行的。通过使用特异性抗体，随后进行¹²⁵I-蛋白A和放射自显影，检测红细胞葡萄糖转运蛋白（一种整合膜蛋白），证明了转移的有效性。本研究使用了几种去污剂，包括辛基葡糖苷、Triton X-100和CHAPS，但只有辛基葡糖苷能有效溶解葡萄糖转运蛋白，且不干扰该蛋白的电转移。在等电聚焦凝胶上分离的葡萄糖转运蛋白在电印迹2小时后被有效转移，其表观pI为6.4 - 6.5。在存在或不存在D-葡萄糖（其抑制[³H]细胞松弛素B与葡萄糖转运蛋白的结合）的情况下，用[³H]细胞松弛素B对红细胞膜进行光标记，并通过双向电泳分离标记的蛋白质，这些发现得到了证实。通过该方法，我们鉴定出一种对D-葡萄糖敏感的50 - 60 kDa蛋白质，其聚焦的表观pI约为pH 6.4 - 6.5。
