Cold Spring Harb Protoc. 2020 Jun 1;2020(6):098426. doi: 10.1101/pdb.prot098426.
Immunoprecipitated proteins can be readily analyzed by immunoblotting. Proteins can be efficiently eluted from the Protein A or similar beads by addition of the SDS-PAGE sample loading buffer and heating at 95°C. This elution procedure will also remove the capturing antibody from the beads unless the antibody was cross-linked to the beads. Alternatively, the immunoprecipitated proteins as well as non-cross-linked capture antibodies can be eluted from the beads using low (2.1-2.8) or high (10-11) pH conditions. Incubation of the immunoprecipitates with the excess of the competing peptide allows the elution of the captured proteins without contamination of the sample with the antibodies present in the immunoprecipitates. However, this option is not always available, and the cost of competing peptide can be prohibitive for the routine immunoprecipitation/immunoblotting experiments. In this protocol, elution of the immunoprecipitated proteins from the beads is performed by mixing Protein A or similar beads containing the immunoprecipitated protein antigens of interest with SDS-PAGE sample buffer and boiling to prepare samples for protein gel electrophoresis.
免疫沉淀的蛋白质可以通过免疫印迹进行分析。通过添加 SDS-PAGE 上样缓冲液并在 95°C 下加热，蛋白质可以从 Protein A 或类似的珠子上有效地洗脱出来。除非抗体与珠子交联，否则该洗脱程序还会将捕获抗体从珠子上洗脱下来。或者，也可以使用低（2.1-2.8）或高（10-11）pH 值条件从珠子上洗脱免疫沉淀的蛋白质和非交联的捕获抗体。将免疫沉淀物与过量的竞争肽孵育，可以在不使存在于免疫沉淀物中的抗体污染样品的情况下洗脱捕获的蛋白质。但是，并非总是可以选择此选项，并且竞争肽的成本可能会使常规免疫沉淀/免疫印迹实验变得昂贵。在本方案中，通过将含有感兴趣的免疫沉淀蛋白抗原的 Protein A 或类似珠子与 SDS-PAGE 上样缓冲液混合并煮沸来从珠子上洗脱免疫沉淀的蛋白质，从而制备用于蛋白质凝胶电泳的样品。
