Purification and Characterization of Clathrin from Bovine Brain

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Abstract
Clathrin was purified to electrophoretic homogeneity by initial extraction to clathrin from bovine brain 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 was obtained. Two forms of native clathrin, fast and slow components, were prepared to about 95% purity by hydroxylapatite column chromatography. Both fast and slow components are believed to represent 2 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 MW of 175,000. 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, 2 proteins of about 38,000 and 35,000 MW that consistently copurified with native clathrin are probably also intrinsic to coated vesicle.