A high-throughput differential filtration assay to screen and select detergents for membrane proteins

• Published in: Analytical biochemistry 2010-12, Vol.407 (1), p.1-11
• Main Authors: Vergis, James M., Purdy, Michael D., Wiener, Michael C.
• Format: Article
• Language: English
• Subjects: Aquaporins - chemistry; Bacterial Proteins - chemistry; Crystallization; Detergents - chemistry; Escherichia coli Proteins - chemistry; Filtration - methods; High-throughput assay; Histidine - chemistry; Histidine - immunology; Membrane protein detergent stability; Membrane Proteins - chemistry; Oligopeptides - chemistry; Oligopeptides - immunology; Potassium Channels - chemistry; Precrystallization screening; Protein Binding; Protein Stability; Recombinant Proteins - chemistry; Solubility; Structural biology
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2010.07.019

Structural studies on integral membrane proteins are routinely performed on protein–detergent complexes (PDCs) consisting of purified protein solubilized in a particular detergent. Of all the membrane protein crystal structures solved to date, a subset of only four detergents has been used in more than half of these structures. Unfortunately, many membrane proteins are not well behaved in these four detergents and/or fail to yield well-diffracting crystals. Identification of detergents that maintain the solubility and stability of a membrane protein is a critical step and can be a lengthy and “protein-expensive” process. We have developed an assay that characterizes the stability and size of membrane proteins exchanged into a panel of 94 commercially available and chemically diverse detergents. This differential filtration assay (DFA), using a set of filtered microplates, requires sub-milligram quantities of purified protein and small quantities of detergents and other reagents and is performed in its entirety in several hours.