Wunder Christian, Lippincott-Schwartz Jennifer, Lorenz Holger
National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA.
Curr Protoc Cell Biol. 2010 Dec;Chapter 5:Unit 5.7. doi: 10.1002/0471143030.cb0507s49.
Correct localization and topology are crucial for a protein's cellular function. To determine topologies of membrane proteins, a new technique, called fluorescence protease protection (FPP) assay, has recently been established. The sole requirements for FPP are the expression of fluorescent-protein fusion proteins and the selective permeabilization of the plasma membrane, permitting a wide range of cell types and organelles to be investigated. Proteins topologies in organelles like endoplasmic reticulum, Golgi apparatus, mitochondria, peroxisomes, and autophagosomes have already been determined by FPP. Here, two different step-by-step protocols of the FPP assay are provided. First, we describe the FPP assay using fluorescence microscopy for single adherent cells, and second, we outline the FPP assay for high-throughput screening applications.
正确的定位和拓扑结构对于蛋白质的细胞功能至关重要。为了确定膜蛋白的拓扑结构，最近建立了一种名为荧光蛋白酶保护（FPP）测定的新技术。FPP的唯一要求是荧光蛋白融合蛋白的表达和质膜的选择性通透，这使得可以研究广泛的细胞类型和细胞器。内质网、高尔基体、线粒体、过氧化物酶体和自噬体等细胞器中的蛋白质拓扑结构已经通过FPP确定。在这里，提供了两种不同的FPP测定分步方案。首先，我们描述了使用荧光显微镜对单个贴壁细胞进行FPP测定，其次，我们概述了用于高通量筛选应用的FPP测定。
