Yagi S, Nakagawa T, Tanaka H, Yoshitake A, Mohri A
Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
J Chromatogr. 1987 Jul 29;400:279-83. doi: 10.1016/s0021-9673(01)81622-8.
A system consisting of high-performance affinity chromatography and size-exclusion chromatography has been developed for the rapid purification and isolation of relatively labile membrane proteins, such as growth-hormone receptor. The crude membrane sample containing growth-hormone receptor was obtained from rabbit livers by ultracentrifugation, followed by solubilization with Triton X-100. The sample was introduced into the high-performance affinity gel column through a large-volume loop injector. After removal of unretained proteins, the fraction containing the growth-hormone receptor was eluted with 6 M urea solution. The eluate from the affinity column passed directly into the size-exclusion gel column, where rapid desalting and separation from contaminants were achieved. The eluate was monitored by UV absorption at 280 nm and fractionated for the subsequent binding assay with 125I-labelled human growth hormone.
已开发出一种由高效亲和色谱和尺寸排阻色谱组成的系统，用于快速纯化和分离相对不稳定的膜蛋白，如生长激素受体。通过超速离心从兔肝脏中获得含有生长激素受体的粗膜样品，然后用 Triton X-100 进行增溶。样品通过大容量定量环进样器引入高效亲和凝胶柱。去除未保留的蛋白质后，用 6 M 尿素溶液洗脱含有生长激素受体的馏分。亲和柱的洗脱液直接进入尺寸排阻凝胶柱，在那里实现了快速脱盐和与污染物的分离。通过在 280 nm 处的紫外吸收监测洗脱液，并进行分馏，以便随后与 125I 标记的人生长激素进行结合测定。
