THE ERASABLE WESTERN-BLOT
THE ERASABLE WESTERN-BLOT
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DOI：
10.1016/0003-2697(87)90656-7
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发表时间：
1987-02-15
影响因子：
2.9
通讯作者：
SHAPER, JH
中科院分区：
文献类型：
--
作者：
KAUFMANN, SH;EWING, CM;SHAPER, JH
开发了一种从硝酸纤维素印迹中成功去除一抗和二抗同时保留最初固定的多肽的方法。在十二烷基硫酸钠存在下通过聚丙烯酰胺凝胶电泳分离多肽，并将其电泳转移到硝酸纤维素上。用5%（w/v）脱脂奶粉封闭非特异性结合位点。在印迹依次与针对目标抗原的抗体和放射性标记的二抗反应后，在干燥和放射自显影之前，需要在5%（w/v）牛奶中洗涤10分钟。在70 ℃孵育30分钟。C在2%（w/v）十二烷基硫酸钠中的溶液，其含有100 mM β-巯基乙醇定量除去抗体并允许重复使用印迹。当蛋白质固定在尼龙上时，这种方法的修改同样允许重复使用Western印迹。讨论了该方法的潜在应用和启示。
A method for successfully removing primary and secondary antibodies from nitrocellulose blots while preserving the originally immobilized polypeptides was developed. Polypeptides were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and electrophoretically transferred to nitrocellulose. Nonspecific binding sites were blocked with 5% (w/v) nonfat dried milk. After blots were reacted sequentially with antibodies directed against the antigen of interest and with radiolabeled secondary antibody, a 10-min wash in 5% (w/v) milk was required prior to drying and autoradiography. A 30-min incubation at 70.degree. C in 2% (w/v) sodium dodecyl sulfate containing 100 mM .beta.-mercaptoethanol quantitatively removed the antibodies and allowed reuse of the blot. A modification of this method similarly allowed reuse of Western blots when proteins were immobilized on nylon. Potential applications and illuminations of this method are discussed.