Moore Priscilla A, Kery Vladimir
Molecular Biosciences, Pacific Northwest National Laboratory, Richland, WA, USA.
Methods Mol Biol. 2009;498:309-14. doi: 10.1007/978-1-59745-196-3_20.
High-throughput protein purification is a complex, multi-step process. There are several technical challenges in the course of this process that are not experienced when purifying a single protein. Among the most challenging are the high-throughput protein concentration and buffer exchange, which are not only labor-intensive but can also result in significant losses of purified proteins. We describe two methods of high-throughput protein concentration and buffer exchange: one using ammonium sulfate precipitation and one using micro-concentrating devices based on membrane ultrafiltration. We evaluated the efficiency of both methods on a set of 18 randomly selected purified proteins from Shewanella oneidensis. While both methods provide similar yield and efficiency, the ammonium sulfate precipitation is much less labor intensive and time consuming than the ultrafiltration.
高通量蛋白质纯化是一个复杂的多步骤过程。在此过程中存在若干技术挑战，而纯化单一蛋白质时不会遇到这些挑战。其中最具挑战性的是高通量蛋白质浓缩和缓冲液交换，这不仅劳动强度大，还可能导致纯化蛋白质的大量损失。我们描述了两种高通量蛋白质浓缩和缓冲液交换方法：一种使用硫酸铵沉淀，另一种使用基于膜超滤的微量浓缩装置。我们对从希瓦氏菌中随机选择的18种纯化蛋白质评估了这两种方法的效率。虽然两种方法的产量和效率相似，但硫酸铵沉淀比超滤的劳动强度和耗时要少得多。
