Brown Austin L, Johnson Brandon E, Goodman Miriam B
Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA.
J Vis Exp. 2008 Oct 16(20):936. doi: 10.3791/936.
Since its development by Sakmann and Neher (1, 2), the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.
自从萨克曼和内尔开发出膜片钳技术(1, 2)以来,它已成为一种极其有用的技术,用于对细胞中的单个或多个离子通道进行电生理测量。该技术可应用于处于天然环境以及在异源细胞(如从非洲爪蟾非洲爪蟾采集的卵母细胞)中表达的离子通道。在此,我们描述从非洲爪蟾卵母细胞进行膜片钳记录的成熟技术。该技术用于测量群体(巨膜片)或单个(单通道记录)表达的离子通道的特性。我们专注于优化卵母细胞制备和封接形成质量的技术。在所有因素都得到优化的情况下,该技术成功形成封接的概率超过90%。每个研究人员可能会根据他或她认为最重要的因素以不同方式优化该过程,我们介绍在我们手中取得最大成功的方法。