Garbern J Y, Wu J Y
J Neurochem. 1981 Feb;36(2):602-12. doi: 10.1111/j.1471-4159.1981.tb01633.x.
Clathrin has been purified to electrophoretic homogeneity by initial extraction of clathrin from purified coated vesicle fraction, followed by column chromatographies with gel filtration, DEAE-cellulose, and hydroxylapatite and finally by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Antibody specific to clathrin has also been obtained. Two forms of native clathrin, fast and slow components, have been prepared to about 95% purity by hydroxylapatite column chromatography. Both fast and slow components are believed to represent two different aggregates of clathrin subunit because they comigrate in agarose electrophoresis, pH 7.4, and also migrate as clathrin subunit on SDS-PAGE with a molecular weight of 175,000. Furthermore, both components cross-react with antibody against purified clathrin and compete for antibody binding site with labeled fast component. The fast component can also be converted to the slow component. In addition to clathrin, two proteins of about 38,000 and 35,000 M.W. that consistently copurified with native clathrin are probably also intrinsic to coated vesicle.
通过从纯化的被膜小泡组分中初步提取网格蛋白，然后依次进行凝胶过滤、二乙氨基乙基纤维素（DEAE -纤维素）和羟基磷灰石柱色谱，最后进行制备性十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS - PAGE），网格蛋白已被纯化至电泳纯。针对网格蛋白的特异性抗体也已获得。通过羟基磷灰石柱色谱已将两种天然形式的网格蛋白，即快速组分和慢速组分，制备至约95%的纯度。快速组分和慢速组分都被认为代表网格蛋白亚基的两种不同聚集体，因为它们在pH值为7.4的琼脂糖电泳中迁移率相同，并且在SDS - PAGE上作为分子量为175,000的网格蛋白亚基迁移。此外，两种组分都能与抗纯化网格蛋白的抗体发生交叉反应，并与标记的快速组分竞争抗体结合位点。快速组分也可以转化为慢速组分。除了网格蛋白外，两种分子量约为38,000和35,000的蛋白质始终与天然网格蛋白共纯化，它们可能也是被膜小泡所固有的。
