Preparation of Plasma Membrane Vesicles, Lysosomes, Proteoliposomes and Alternative Sample Types for SSM-Based...

Solid-supported membrane-based electrophysiology (SSME) is a vital technique for characterizing electrogenic membrane protein activity, with reliable measurements critically dependent on high-quality sample preparation. This chapter presents a comprehensive, step-by-step protocol for the purification of plasma membrane vesicles and lysosomes from mammalian cell culture, specifically optimized for SSME recordings. The method combines nitrogen decompression with discontinuous sucrose gradient centrifugation to maximize purity and yield, thereby substantially improving the signal-to-noise ratio compared to crude membrane extracts. To evaluate the purity of the resulting membrane fractions, we analyzed the enrichment of 14 marker proteins representing not only plasma membrane and lysosomes, but also endosomes, endoplasmic reticulum, Golgi apparatus, mitochondria, and other organelles. In addition, the chapter provides guidance on adapting the protocol to different experimental requirements, including alternative cell disruption techniques, approaches for enriching other organelles, and the use of crude membrane preparations in high-throughput screening applications. Alternative sample sources and preparation strategies are also discussed, encompassing bacterial membranes, native tissues, cell-free expression systems, and intact cultured cells, with references to established protocols. A dedicated section describes the generation of proteoliposomes-an important complementary sample type that offers greater flexibility for studying low-turnover transporters and for achieving precise control of membrane composition. Collectively, this methodological framework serves as a basis for SSME sample preparation and underscores the versatility of SSME.