Analysis of Protein and Lipid Dynamics Using Confocal Fluorescence Recovery After Photobleaching

Fluorescence recovery after photobleaching (FRAP) is a powerful, versatile, and widely accessible tool to monitor molecular dynamics in living cells that can be performed using modern confocal microscopes. Although the basic principles of FRAP are simple, quantitative FRAP analysis requires careful experimental design, data collection, and analysis. In this article, we discuss the theoretical basis for confocal FRAP, followed by step-by-step protocols for FRAP data acquisition using a laser-scanning confocal microscope for (1) measuring the diffusion of a membrane protein, (2) measuring the diffusion of a soluble protein, and (3) analyzing intracellular trafficking. Finally, data analysis procedures are discussed, and an equation for determining the diffusion coefficient of a molecular species undergoing pure diffusion is presented. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: How to set up a FRAP experiment Basic Protocol 2: Confocal FRAP measurements of the lateral diffusion of plasma membrane proteins and lipids Alternate Protocol 1: Lateral diffusion measurements for a rapidly diffusing soluble protein Alternate Protocol 2: FRAP analysis of intracellular trafficking kinetics Basic Protocol 3: Working with FRAP data Basic Protocol 4: Further analysis of FRAP data to obtain diffusion coefficients.