St. Jude Children's Research Hospital, Memphis, Tennessee.
Curr Protoc. 2022 Sep;2(9):e546. doi: 10.1002/cpz1.546.
Expressing recombinant proteins in heterologous host cells is a prerequisite for purification and other downstream processes. Cell cultures require a protein expression test to optimize incubation time, temperature, and additives (like chemical inducers) to identify the best growth conditions with maximum recombinant protein yield. However, running SDS-PAGE followed by western blotting is cumbersome and results are not quick. Here, I describe a simple protocol to quickly check the presence of recombinant protein in cell cultures using a dot-blot experiment. The cells can be rapidly lysed and directly spotted on the nitrocellulose membrane. Then, the membrane is incubated with a horseradish peroxidase (HRP) conjugated antibody raised against the affinity tag present on the recombinant protein to confirm the protein expression by chemiluminescence. It takes less than an hour to get results. This method rapidly investigates recombinant protein expression in different cell lines and tests other variables. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Protein expression analysis for eukaryotic systems Basic Protocol 2: Protein expression analysis for bacterial systems.
在异源宿主细胞中表达重组蛋白是纯化和其他下游处理的前提条件。细胞培养需要进行蛋白表达测试，以优化孵育时间、温度和添加物（如化学诱导剂），从而确定最佳生长条件和最大重组蛋白产量。然而，进行 SDS-PAGE 凝胶电泳和随后的 Western blot 实验繁琐，结果也不够快速。本文描述了一种简单的方案，使用点印迹实验快速检查细胞培养物中重组蛋白的存在。可以快速裂解细胞，并将其直接点样到硝酸纤维素膜上。然后，将膜与针对重组蛋白上亲和标签的辣根过氧化物酶（HRP）偶联抗体孵育，通过化学发光法确认蛋白表达。不到一个小时即可得到结果。该方法可快速研究不同细胞系中的重组蛋白表达，并测试其他变量。© 2022 威立出版公司。 基本方案 1：真核系统的蛋白表达分析 基本方案 2：细菌系统的蛋白表达分析。
