释义：

Proteins are fractionated by PAGE (SDS-polyacrylamide gel electrophoresis), transferred to a nitrocellulose membrane and tested for the ability to bind to a protein, a peptide, or any other ligand. Cell lysates can also be fractionated before gel electrophoresis to increase the sensitivity of the method for detecting interactions with rare proteins. Denaturants are removed during the blotting procedure, which allows many proteins to recover (or partially recover) activity. However, if biological activity is not recoverable, the proteins can be fractionated by a non denaturing gel system. This variation of the method eliminates the problem of activity regeneration and allows the detection of binding when the presence of a protein complex is required for binding. The protein probe can be prepared by any one of several procedures, while fusion affinity tags greatly facilitate purification. Synthesis in E. coli with a GST fusion, epitope tag, or other affinity tag is most commonly used. The protein of interest can then be radioactively labelled, biotinylated, or used in the blotting procedure as an unlabeled probe that is detected by a specific antibody.

蛋白质通过 PAGE (SDS-聚丙烯酰胺凝胶电泳) 进行分离，转移到硝酸纤维素膜上，并测试其与蛋白质、肽或其他配体的结合能力。细胞裂解液也可以在凝胶电泳之前进行分离，以提高检测稀有蛋白质相互作用的灵敏度。在 blotting 过程中去除变性剂，这使许多蛋白质可以恢复 (或部分恢复) 活性。然而，如果生物活性无法恢复，可以通过非变性凝胶系统对蛋白质进行分离。这种方法的变体消除了活性再生的问题，并允许在需要蛋白质复合物存在时检测结合。蛋白质探针可以通过多种方法准备，而融合亲和标签大大促进了纯化。最常用的方法是在大肠杆菌中合成 GST 融合、表位标签或其他亲和标签。然后，感兴趣的蛋白质可以进行放射性标记、偶联生物素或在 blotting 过程中作为未标记探针，使用特定抗体进行检测。