Cold Spring Harb Protoc. 2021 Dec 1;2021(12):2021/12/pdb.prot102251. doi: 10.1101/pdb.prot102251.
In immunoblotting (western blotting), proteins are first separated by SDS-PAGE and then transferred electrophoretically from the gel onto a support membrane that binds proteins tightly. After the unreacted binding sites of the membrane are blocked to suppress nonspecific adsorption of antibodies, the immobilized proteins are reacted with a specific polyclonal or monoclonal antibody. Antigen-antibody complexes are visualized using chromogenic, fluorescent, or chemiluminescent reactions. Immunoblotting protocols are reagent specific and, owing to the wide assortment of equipment, reagents, and antibodies available, highly diverse. Presented here is an example of a workable protocol for developing a blot using horseradish peroxidase (HRP)-conjugated secondary antibody and enhanced chemiluminescence (ECL). ECL is based on the emission of light during the HRP-catalyzed oxidation of luminal or other substrates. Emitted light is captured on film or by a CCD camera, for qualitative or semiquantitative analysis. Because ECL is so sensitive, it has become a popular detection method. This protocol can be modified for different membranes, antibodies, and detection systems. Optimal dilutions of the primary and secondary antibodies need to be determined empirically, but recommendations provided by the manufacturer are usually a good starting point.
在免疫印迹（western blot）中，蛋白质首先通过 SDS-PAGE 分离，然后通过电泳从凝胶转移到紧密结合蛋白质的支持膜上。在阻断膜上未反应的结合位点以抑制抗体的非特异性吸附后，将固定化的蛋白质与特异性的多克隆或单克隆抗体反应。使用显色、荧光或化学发光反应来可视化抗原-抗体复合物。免疫印迹方案是试剂特异性的，并且由于可用的设备、试剂和抗体种类繁多，因此高度多样化。这里提供了一个使用辣根过氧化物酶（HRP）缀合的二级抗体和增强化学发光（ECL）开发印迹的可行方案示例。ECL 基于 HRP 催化的内腔或其他底物氧化期间光的发射。发射的光被捕获在胶片上或 CCD 相机上，用于定性或半定量分析。由于 ECL 非常灵敏，它已成为一种流行的检测方法。此方案可针对不同的膜、抗体和检测系统进行修改。需要通过经验确定一抗和二抗的最佳稀释度，但制造商提供的建议通常是一个很好的起点。
