Wiesler Simone C, Weinzierl Robert O J
Department of Life Sciences, Imperial College London, Sir Alexander Fleming Building, Exhibition Road, London, SW7 2AZ, UK.
Methods Mol Biol. 2015;1286:97-106. doi: 10.1007/978-1-4939-2447-9_9.
Affinity purification of recombinant proteins has become the method of choice to obtain good quantities and qualities of proteins for a variety of downstream biochemical applications. While manual or FPLC-assisted purification techniques are generally time-consuming and labor-intensive, the advent of high-throughput technologies and liquid handling robotics has simplified and accelerated this process significantly. Additionally, without the human factor as a potential source of error, automated purification protocols allow for the generation of large numbers of proteins simultaneously and under directly comparable conditions. The delivered material is ideal for activity comparisons of different variants of the same protein. Here, we present our strategy for the simultaneous purification of up to 24 affinity-tagged proteins for activity measurements in biochemical assays. The protocol described is suitable for the scale typically required in individual research laboratories.
重组蛋白的亲和纯化已成为为各种下游生化应用获取大量优质蛋白的首选方法。虽然手动或FPLC辅助纯化技术通常耗时且费力，但高通量技术和液体处理机器人技术的出现显著简化并加速了这一过程。此外，由于不存在人为因素这一潜在误差来源，自动化纯化方案能够在直接可比的条件下同时生成大量蛋白质。所提供的材料非常适合用于同一蛋白质不同变体的活性比较。在此，我们展示了我们同时纯化多达24种亲和标签蛋白以用于生化分析中活性测量的策略。所描述的方案适用于单个研究实验室通常所需的规模。
