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

Abstract

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.

The monomeric state of the proton‐coupled folate transporter represents the functional unit in the plasma membrane

“…To this end we used Jacobson's pellicle method [46]. Intact cells (oocytes or CHO) were treated with cationic silica to coat the exterior of the plasma membranes [47,48]. The colloidal silica particles were then crosslinked by polyacrylic acid.…”

Role of multiple phosphorylation sites in the COOH-terminal tail of aquaporin-2 for water transport: evidence against channel gating

“…Oocyte membranes were purified using a modification of a technique described by Leduc-Nadeau et al (18). In brief, for the preparation of total membranes, five oocytes were homogenized in 1 ml buffer (HbAϩ: 5 mM MgCl2, 5 mM NaH2PO4, 1 mM EDTA, 80 mM sucrose, 20 mM Tris, pH 7.48, and 8 M leupeptin, and 0.4 mM Pefabloc).…”

Exogenous application of abscisic acid (ABA) increases root and cell hydraulic conductivity and abundance of some aquaporin isoforms in the ABA-deficient barley mutant Az34

“…Briefly, the coding region of HvPIP2;5 cDNAs was sub-cloned into pXbG-ev1, and corresponding cRNA was synthesized and injected into oocytes. Total membranes of oocytes expressing HvPIP2;5 protein were extracted according to Leduc-Nadeau et al (2007). All membrane protein corresponding to one oocyte was used as a sample and subjected to solubilization, SDS-PAGE and Western blotting as described previously (Katsuhara et al, 2002).…”