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1 Physiology, university of Montreal, montreal, Canada
2 Physiology, university of Montreal, Montreal, Canada
3 de Physique, University of Montreal, Montreal, Canada
4 Centre de recherche, Hopital du Sacre-Coeur de Montreal, Montreal H4J 1C5, Canada
* To whom correspondence should be addressed. E-mail: pierre.bissonnette{at}umontreal.ca.
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 implication of mistargeting in relation to the pathogenesis of to these proteins. In order 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 paper, we present a new technique which permits excellent purification of plasma membranes from Xenopus laevis oocytes. This technique is fast, does not require particular skill, such as peeling of vitelline membrane, and permits purification of multiple samples from as few as ten and up to more than a hundred oocytes. The procedure combines partial digestion of the vitelline membrane, polymerization of the plasma membrane and low speed centrifugations. We have validated this technique using essentially Western blots assays on three plasma membrane proteins (AQP2, SGLT1 and TRPV5) 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.
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