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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes

¹Groupe d’Étude des Protéines Membranaires, Département de Physiologie, Université de Montréal, and ²Centre de Recherche, Hôpital du Sacré-Cur de Montréal, Montreal, Quebec, Canada

Submitted 24 March 2006 ; accepted in final form 29 October 2006

Over the past two decades, Xenopus laevis oocytes have been widely used as an expression system to investigate both physiological and pathological properties of membrane proteins such as channels and transporters. Past studies have clearly shown the key implications of mistargeting in relation to the pathogenesis of these proteins. To unambiguously determine the plasma membrane targeting of a protein, a thorough purification technique becomes essential. Unfortunately, available techniques are either too cumbersome, technically demanding, or require large amounts of material, all of which are not adequate when using oocytes individually injected with cRNA or DNA. In this article, we present a new technique that permits excellent purification of plasma membranes from X. laevis oocytes. This technique is fast, does not require particular skills such as peeling of vitelline membrane, and permits purification of multiple samples from as few as 10 and up to >100 oocytes. The procedure combines partial digestion of the vitelline membrane, polymerization of the plasma membrane, and low-speed centrifugations. We have validated this technique essentially with Western blot assays on three plasma membrane proteins [aquaporin (AQP)2, Na⁺-glucose cotransporter (SGLT)1, and transient receptor potential vanilloid (TRPV)5], using both wild-type and mistargeted forms of the proteins. Purified plasma membrane fractions were easily collected, and samples were found to be adequate for Western blot identification.

expression studies; aquaporin 2 mutations