Unit

UNIT 2.19 Analysis of Protein and Lipid Dynamics Using Confocal Fluorescence Recovery After Photobleaching (FRAP)

  1. Charles A. Day1,4,
  2. Lewis J. Kraft2,4,
  3. Minchul Kang1,4,
  4. Anne K. Kenworthy1,2,3

Published Online: 1 OCT 2012

DOI: 10.1002/0471142956.cy0219s62

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Day, C. A., Kraft, L. J., Kang, M. and Kenworthy, A. K. 2012. Analysis of Protein and Lipid Dynamics Using Confocal Fluorescence Recovery After Photobleaching (FRAP). Current Protocols in Cytometry. 62:2.19:2.19.1–2.19.29.

Author Information

  1. 1

    Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee

  2. 2

    Chemical and Physical Biology Program, Vanderbilt University School of Medicine, Nashville, Tennessee

  3. 3

    Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee

  4. 4

    These authors contributed equally to this work.

Publication History

  1. Published Online: 1 OCT 2012
  2. Published Print: OCT 2012

Abstract

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 unit, 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) analysis of 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. Curr. Protoc. Cytom. 62:2.19.1-2.19.29. © 2012 by John Wiley & Sons, Inc.

Keywords:

  • FRAP;
  • diffusion;
  • confocal laser-scanning microscopes;
  • protein trafficking;
  • fluorescence microscopy;
  • GFP