Gallagher Sean, Winston Scott E, Fuller Steven A, Hurrell John G R
UVP Inc, Upland, California, USA.
Curr Protoc Neurosci. 2004 Nov;Chapter 5:Unit 5.19. doi: 10.1002/0471142301.ns0519s29.
Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides numerous protocols for all steps starting with solubilization of the protein samples, usually with SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are then electrophoretically transferred to a membrane, a process that can be monitored by reversible staining or Ponceau S staining. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. Any remaining binding sites are blocked by immersing the membrane in a blocking solution. After probing with the primary antibody, the membrane is washed and the antibody-antigen complexes are identified with horseradish peroxidase (HRPO) or alkaline phosphatase enzymes coupled to the secondary anti-IgG antibody (e.g., goat anti-rabbit IgG) and 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。本单元提供了从蛋白质样品溶解开始的所有步骤的众多方案，通常使用SDS和还原剂。溶解后，材料通过SDS-PAGE分离，然后将抗原电泳转移到膜上，这一过程可通过可逆染色或丽春红S染色进行监测。转移的蛋白质结合到膜表面，便于使用免疫检测试剂。通过将膜浸入封闭溶液中来封闭任何剩余的结合位点。用一抗探测后，洗涤膜，并用与二抗IgG抗体（如山羊抗兔IgG）偶联的辣根过氧化物酶（HRPO）或碱性磷酸酶以及适当的显色或发光底物来鉴定抗体-抗原复合物。最后，膜可以被剥离并重新探测。
