Towbin H, Staehelin T, Gordon J
Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4. doi: 10.1073/pnas.76.9.4350.
A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
已设计出一种将蛋白质从聚丙烯酰胺凝胶电泳转移至硝酸纤维素膜的方法。该方法可实现从含尿素的凝胶中定量转移核糖体蛋白。对于十二烷基硫酸钠凝胶，能获得原始条带模式且分辨率无损失，但转移并非定量的。此方法可通过放射自显影检测蛋白质，且比传统方法更简便。固定化蛋白质可通过免疫程序检测。用过量蛋白质封闭硝酸纤维素膜上所有额外的结合能力；然后结合特异性抗体，最后结合针对第一抗体的第二抗体。第二抗体要么用放射性标记，要么与荧光素或过氧化物酶偶联。然后分别通过放射自显影、在紫外光下或通过过氧化物酶反应产物检测特异性蛋白质。在后一种情况下，低至100 pg的蛋白质也能清晰检测到。预计该程序将适用于分析具有特异性反应或配体的多种蛋白质。
