The fluorescence protease protection (FPP) assay to determine protein localization and membrane topology
The fluorescence protease protection (FPP) assay to determine protein localization and membrane topology
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DOI：
10.1038/nprot.2006.42
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Lippincott-Schwartz, Jennifer
中科院分区：
文献类型：
--
作者：
Lorenz, Holger;Hailey, Dale W.;Lippincott-Schwartz, Jennifer
正确的定位和拓扑结构对蛋白质的细胞功能至关重要。为了确定膜蛋白的拓扑结构，可以应用一种称为荧光蛋白酶保护（FPP）试验的新技术。该试验使用GFP标记的跨膜蛋白的受限蛋白水解消化率来指示其膜内取向。FPP的唯一要求是GFP融合蛋白的表达和质膜的选择性渗透，这允许广泛的细胞类型和细胞器进行研究。FPP测定可以直接进行，在几分钟内获得可靠的结果。在这里，我们提供了一个一步一步的实验方案。作为一个例子，我们使用FPP来确定内质网（ER）跨膜蛋白的哪个端是管状的，哪个端是面向细胞质溶胶的。
Correct localization and topology are crucial for the cellular function of a protein. To determine the topology of membrane proteins, a new technique, called the fluorescence protease protection ( FPP) assay, can be applied. This assay uses the restricted proteolytic digestibility of GFP- tagged transmembrane proteins to indicate their intramembrane orientation. The sole requirements for FPP are the expression of GFP fusion proteins and the selective permeabilization of the plasma membrane, which permits a wide range of cell types and organelles to be investigated. The FPP assay can be carried out in a straightforward manner to obtain reliable results within minutes. Here we provide a step- by- step protocol for the assay. As an example, we use FPP to determine which terminus of an endoplasmic reticulum ( ER) transmembrane protein is lumenal and which one is facing the cytosol.