Abteilung für Systembiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, Bochum, Germany.
Institut für Biochemie und Pathobiochemie, Ruhr-Universität Bochum, Bochum, Germany.
Methods Mol Biol. 2023;2643:373-382. doi: 10.1007/978-1-0716-3048-8_26.
For a long time, the isolation of native protein complexes from human cells was accomplished by immunoprecipitation experiments. However, success depends on the quality of the antibodies and the method consumes valuable antibodies, which can hinder subsequent analysis of the isolated complexes. Here, we demonstrate an alternative approach based on affinity purification. It utilizes human Flp-In cells, which genomically express a Protein A-tagged version of the human peroxisomal import receptor PEX5L. Native soluble and membrane-bound complexes containing PEX5L can thereby be isolated via a well-known affinity-based strategy.
长期以来，从人类细胞中分离天然蛋白复合物都是通过免疫沉淀实验来完成的。然而，这种方法的成功与否取决于抗体的质量，而且该方法还会消耗宝贵的抗体，这可能会阻碍对分离出的复合物的后续分析。在这里，我们展示了一种基于亲和纯化的替代方法。它利用了人类 Flp-In 细胞，这些细胞在基因组上表达了一种带有蛋白 A 标签的人过氧化物酶体输入受体 PEX5L。因此，通过一种众所周知的基于亲和力的策略，可以分离含有 PEX5L 的天然可溶性和膜结合复合物。
