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首页> 外文期刊>American Journal of Physiology >Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes.
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Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes.

机译：详细阐述了从非洲爪蟾卵母细胞中纯化质膜的新技术。

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摘要

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.
机译：在过去的二十年中，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。过去的研究清楚地表明了与这些蛋白质的发病机制有关的靶向错误的关键含义。为了明确确定蛋白质的质膜靶向性，彻底的纯化技术变得至关重要。不幸的是，可用的技术要么太麻烦，技术要求高，要么需要大量的材料，当使用单独注射了cRNA或DNA的卵母细胞时，所有这些都不足够。在本文中，我们提出了一种新技术，该技术可以从X. laevis卵母细胞中出色地纯化质膜。该技术速度快，不需要诸如卵黄膜剥离的特殊技能，并允许从少至10个卵母细胞和多达100个以上的卵母细胞中纯化多个样品。该程序结合了卵黄膜的部分消化，质膜的聚合和低速离心。我们已经使用两种野生型的三种质膜蛋白[aquaporin（AQP）2，Na（+）-葡萄糖共转运蛋白（SGLT）1和瞬时受体电位类香草素（TRPV）5]进行了蛋白质印迹实验，从而验证了该技术的有效性。和蛋白质靶向错误的形式。纯化的质膜级分很容易收集，并且发现样品足以进行蛋白质印迹鉴定。

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