The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA.
Nat Protoc. 2013 Feb;8(2):345-54. doi: 10.1038/nprot.2013.005. Epub 2013 Jan 24.
The distinctive distributions of proteins within subcellular compartments both at steady state and during signaling events have essential roles in cell function. Here we describe a method for delineating the complex arrangement of proteins within subcellular structures visualized using point-localization superresolution (PL-SR) imaging. The approach, called pair correlation photoactivated localization microscopy (PC-PALM), uses a pair-correlation algorithm to precisely identify single molecules in PL-SR imaging data sets, and it is used to decipher quantitative features of protein organization within subcellular compartments, including the existence of protein clusters and the size, density and number of proteins in these clusters. We provide a step-by-step protocol for PC-PALM, illustrating its analysis capability for four plasma membrane proteins tagged with photoactivatable GFP (PAGFP). The experimental steps for PC-PALM can be carried out in 3 d and the analysis can be done in ∼6-8 h. Researchers need to have substantial experience in single-molecule imaging and statistical analysis to conduct the experiments and carry out this analysis.
在稳定状态和信号转导事件中，蛋白质在亚细胞隔室中的独特分布在细胞功能中起着重要作用。在这里，我们描述了一种用于描绘使用点定位超分辨率（PL-SR）成像可视化的亚细胞结构内复杂蛋白质排列的方法。该方法称为对相关光激活定位显微镜（PC-PALM），它使用对相关算法来精确识别 PL-SR 成像数据集内的单个分子，并用于破译亚细胞隔室内蛋白质组织的定量特征，包括蛋白质簇的存在以及这些簇中的蛋白质的大小、密度和数量。我们提供了 PC-PALM 的分步协议，说明了其对用光活化 GFP（PAGFP）标记的四种质膜蛋白的分析能力。PC-PALM 的实验步骤可以在 3 天内完成，分析可以在 6-8 小时内完成。研究人员需要在单分子成像和统计分析方面有丰富的经验才能进行实验和进行此分析。
