下载PDF
{"title":"Immunoblotting and Immunodetection.","authors":"Duojiao Ni, Peng Xu, Sean Gallagher","doi":"10.1002/cpps.32","DOIUrl":null,"url":null,"abstract":"<p><p>Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with fluorophores, horseradish peroxidase, or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate fluorescent imaging technologies or with chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed. © 2017 by John Wiley & Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"88 ","pages":"10.10.1-10.10.37"},"PeriodicalIF":0.0000,"publicationDate":"2017-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.32","citationCount":"81","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Protocols in Protein Science","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1002/cpps.32","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 81
引用
批量引用
Abstract
Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with fluorophores, horseradish peroxidase, or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate fluorescent imaging technologies or with chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed. © 2017 by John Wiley & Sons, Inc.
免疫印迹和免疫检测。
免疫印迹法(western blotting)用于鉴定被多克隆或单克隆抗体识别的特异性抗原。该单元提供了所有步骤的协议,从蛋白质样品的溶解开始,通常通过SDS和还原剂。溶解后,材料通过SDS-PAGE分离,抗原通过电泳转移到膜上,这一过程可以通过Ponceau s染色进行监测。转移的蛋白质结合到膜表面,为免疫检测试剂提供了通道。在非特异性结合位点被阻断后,用一抗探测膜并清洗。抗体-抗原复合物用荧光基团、辣根过氧化物酶或碱性磷酸酶偶联到二级抗igg抗体进行标记,并使用适当的荧光成像技术或显色或发光底物进行检测。最后,膜可能被剥离和谴责。©2017 by John Wiley & Sons, Inc。
本文章由计算机程序翻译,如有差异,请以英文原文为准。