Giant liposome preparation for imaging and patch-clamp electrophysiology.
Giant liposome preparation for imaging and patch-clamp electrophysiology.
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DOI：
10.3791/50227
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发表时间：
2013-06-21
期刊：
影响因子：
--
通讯作者：
Gordon SE
中科院分区：
文献类型：
--
作者：
Collins MD;Gordon SE
将离子通道重组到化学定义的脂质膜中用于电生理记录已经成为鉴定和探索这些重要蛋白质功能的有力技术。然而，经典的制备，如平面双层，限制了可以在重构通道及其膜环境上进行的操作和实验。巨型脂质体的更像细胞的结构允许传统的膜片钳实验，而不牺牲对脂质环境的控制。电成型是一种制备直径大于10 μm的巨型脂质体的有效方法，它依赖于在电极表面沉积一层薄的有序脂质膜。然而，由于经典方案要求脂质从有机溶剂中沉积，因此它与不太稳健的膜蛋白如离子通道不相容，必须进行修饰。最近，已经开发出从部分脱水的小脂质体电形成巨脂质体的方案，我们已经在实验室中将其适应于含蛋白质的脂质体。我们在这里介绍的背景，设备，技术，和缺陷的电铸巨脂质体从小脂质体分散体。我们开始与经典的协议，这应该首先掌握，然后再尝试更具挑战性的协议，以下。我们证明了控制的小脂质体的部分脱水的过程中使用饱和盐溶液的蒸汽平衡。最后，我们展示了电铸本身的过程。我们将描述简单，廉价的设备，可以在内部生产高质量的脂质体，并描述在每个阶段的制备目视检查，以确保最佳结果。
The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment. Electroformation is an efficient mean to produce giant liposomes >10 μm in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory. We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.
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影响因子：
3.4
作者：
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3.4
作者：
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作者：
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DOI：
10.1016/s0021-9673(00)89973-2
发表时间：
1978-01-01
期刊：
JOURNAL OF CHROMATOGRAPHY
影响因子：
--
作者：
BAKALYAR, SR;BRADLEY, MPT;HONGANEN, R
通讯作者：
HONGANEN, R
DOI：
10.1016/0006-291x(71)90443-8
发表时间：
1971-01-01
影响因子：
3.1
作者：
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通讯作者：
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