Clear Native Gel Electrophoresis for the Purification of Fluorescently Labeled Membrane Proteins in Native Nanodiscs.

Native gel electrophoresis techniques, such as blue or clear native gel electrophoresis (BNE or CNE), are widely used to separate and characterize proteins. However, in high-resolution CNE, mild anionic or neutral detergents are often used at concentrations that are too low to prevent membrane-protein aggregation. Additionally, the identification of proteins is hampered by the lack of suitable molecular-weight markers such as those used in SDS-PAGE. Here, we introduce a novel approach that combines charged polymer-encapsulated nanodiscs and fluorescence correlation spectroscopy (FCS) to address both challenges. Membrane proteins are first extracted using Glyco-DIBMA, a negatively charged amphiphilic copolymer. This enables the spontaneous formation of nanodiscs harboring the fluorescently labeled target protein within a native-like lipid-bilayer environment, which is confirmed by FCS. The nanodiscs are then subjected to detergent-free CNE. As the number of protomers increases, the nanodiscs grow larger, resulting in increased migration distances in CNE due to higher charge densities. Crucially, the nanodiscs remain intact throughout the CNE, as demonstrated by FCS analysis of resolubilized bands excised from the gels. Moreover, the membrane proteins used in this study, a potassium channel (KvAP), a sodium channel (NavMs), a water channel (GlpF), and a urea channel (