Gallagher Sean, Winston Tank Transfer Systems Scott E, Fuller Tank Transfer Systems Steven A, Hurrell Tank Transfer Systems Reversible Staining Of Proteins John G R
UVP, Inc, Upland, California.
Nabi, Rockville, Maryland.
Curr Protoc Immunol. 2008 Nov;Chapter 8:8.10.1-8.10.28. doi: 10.1002/0471142735.im0810s83.
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 horseradish peroxidase or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.
免疫印迹法（蛋白质免疫印迹法）用于鉴定由多克隆或单克隆抗体识别的特定抗原。本单元提供了所有步骤的方案，从蛋白质样品的溶解开始，通常通过SDS和还原剂来实现。溶解后，材料通过SDS-PAGE进行分离，抗原通过电泳转移到膜上，这一过程可以通过用丽春红S进行可逆染色来监测。转移的蛋白质结合在膜的表面，便于免疫检测试剂与之结合。在封闭非特异性结合位点后，用一抗对膜进行检测并洗涤。抗体-抗原复合物用与二抗IgG偶联的辣根过氧化物酶或碱性磷酸酶进行标记，并使用适当的显色或发光底物进行检测。最后，膜可以进行剥离并重新检测。
