Reiser J, Wardale J
Eur J Biochem. 1981 Mar;114(3):569-75. doi: 10.1111/j.1432-1033.1981.tb05182.x.
We describe a sensitive immunological procedure for the detection of specific proteins in total cell extracts and for the comparison of antigenically related polypeptides. Proteins are fractionated in polyacrylamide gels and transferred electrophoretically to diazophenylthioether paper, to which they bind covalently. Specific proteins are identified by incubation with specific antibody and 125 I-labeled protein A from Staphylococcus aureus, followed by autoradiography. High-resolution separation of proteins prior to transfer is achieved by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate or by nonequilibrium pH gradient electrophoresis, followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Further information can be obtained by limited enzymatic proteolysis of the proteins in the gel following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and analysis of the cleavage products by gel electrophoresis at right angles to the first gel. We show the application of this technique to the detection and comparison in extracts from infected cells of proteins related immunologically to the simian virus 40 capsid proteins VP1 and VP3.
我们描述了一种灵敏的免疫学方法，用于检测全细胞提取物中的特定蛋白质以及比较抗原相关的多肽。蛋白质在聚丙烯酰胺凝胶中进行分级分离，然后通过电泳转移至重氮苯硫醚纸上，并与之共价结合。通过与特异性抗体和来自金黄色葡萄球菌的125I标记蛋白A孵育，随后进行放射自显影来鉴定特定蛋白质。在转移之前，可通过在十二烷基硫酸钠存在下进行聚丙烯酰胺梯度凝胶电泳或非平衡pH梯度电泳，随后在十二烷基硫酸钠存在下进行聚丙烯酰胺凝胶电泳，实现蛋白质的高分辨率分离。在十二烷基硫酸钠存在下进行聚丙烯酰胺凝胶电泳后，通过对凝胶中的蛋白质进行有限的酶促蛋白水解，并通过与第一块凝胶成直角的凝胶电泳分析裂解产物，可获得更多信息。我们展示了该技术在检测和比较感染细胞提取物中与猴病毒40衣壳蛋白VP1和VP3免疫相关的蛋白质方面的应用。
