TRANSFER OF PROTEINS FROM GELS TO DIAZOBENZYLOXYMETHYL-PAPER AND DETECTION WITH ANTISERA - METHOD FOR STUDYING ANTIBODY SPECIFICITY AND ANTIGEN STRUCTURE
TRANSFER OF PROTEINS FROM GELS TO DIAZOBENZYLOXYMETHYL-PAPER AND DETECTION WITH ANTISERA - METHOD FOR STUDYING ANTIBODY SPECIFICITY AND ANTIGEN STRUCTURE
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DOI：
10.1073/pnas.76.7.3116
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发表时间：
1979-01-01
影响因子：
11.1
通讯作者：
STARK, GR
中科院分区：
文献类型：
--
作者：
RENART, J;REISER, J;STARK, GR
本文报道了一种快速、灵敏的检测蛋白质抗原的方法。聚丙烯酰胺基质与试剂交联，该试剂可以用高碘酸盐或碱裂解，以促进蛋白质条带转移到重氮苄氧基甲基纸上，在那里它们共价偶联。用未分级、未标记的特异性抗血清和125 I标记的金黄色葡萄球菌蛋白A依次孵育后，通过放射自显影法检测特异性蛋白。抗体和蛋白A可以用尿素和2-巯基乙醇去除，并且同一张纸可以用不同的抗血清再次探测。制备了SV-40病毒体蛋白VP 3和VP 2的特异性抗血清;如该方法所示，其不与VP 1交叉反应。兔抗SV-40转化兔肾细胞的抗血清主要针对高碘酸盐敏感部分，该部分存在于感染或转化细胞的肿瘤（T）抗原中，而兔抗十二烷基硫酸钠存在下通过电泳从裂解感染的猴肾细胞中纯化的大T抗原的抗血清主要针对对高碘酸盐不敏感的决定簇。
A rapid and very sensitive method was described for detecting proteins as antigens after their separation in polyacrylamide/agarose composite gels, with or without sodium dodecyl sulfate. The polyacrylamide matrix was crosslinked with a reagent that could be cleaved with periodate or alkali to facilitate transfer of the protein bands to diazobenzyloxymethyl-paper, where they were coupled covalently. Specific proteins were detected by autoradiography after sequential incubation with unfractionated, unlabeled specific antiserum and 125I-labeled protein A from Staphylococcus aureus. Antibody and protein A could be removed with urea and 2-mercaptoethanol, and the same paper could be probed again with a different antiserum. An antiserum specific for the SV-40 virion proteins VP3 and VP2 was prepared; it did not crossreact with VP1, as demonstrated by this method. An antiserum raised in rabbits against SV-40-transformed rabbit kidney cells was directed primarily against a periodate-sensitive moiety present in tumor (T) antigen from infected or transformed cells, whereas an antiserum raised in rabbits against large T antigen purified from lytically infected monkey kidney cells by electrophoresis in the presence of sodium dodecyl sulfate was directed primarily against-determinants that were not sensitive to periodate.