Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes
Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes
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DOI：
10.1152/ajpcell.00136.2006
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发表时间：
2007-03-01
影响因子：
5.5
通讯作者：
Bichet, Daniel G.
中科院分区：
文献类型：
--
作者：
Leduc-Nadeau, Alexandre;Lahjouji, Karim;Bichet, Daniel G.
在过去的二十年里，非洲爪蟾卵母细胞已被广泛用作表达系统，以研究膜蛋白，如通道和转运蛋白的生理和病理特性。过去的研究已经清楚地表明了这些蛋白质的发病机制的错误的关键影响。为了明确地确定蛋白质的质膜靶向，彻底的纯化技术变得至关重要。不幸的是，现有的技术要么太麻烦，技术要求高，要么需要大量的材料，当使用单独注射cRNA或DNA的卵母细胞时，所有这些都是不够的。在这篇文章中，我们提出了一种新的技术，允许优秀的纯化质膜从X。卵母细胞该技术是快速的，不需要特殊的技能，如剥离卵黄膜，并允许从少至10个和多达> 100个卵母细胞的多个样品的纯化。该程序结合了卵黄膜的部分消化、质膜的聚合和低速离心。我们已经验证了这一技术基本上与蛋白质印迹分析三个质膜蛋白[水通道蛋白（AQP）2，Na+-葡萄糖协同转运蛋白（SGLT）1，和瞬时受体电位香草酸（TRPV）5]，使用野生型和错误的形式的蛋白质。容易收集纯化的质膜组分，并且发现样品足以用于Western印迹鉴定。
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.