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

Charles A. Day1, Lewis J. Kraft1, Minchul Kang1, Anne K. Kenworthy2

1 These authors contributed equally to this work., 2 Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 2.19
DOI:  10.1002/0471142956.cy0219s62
Online Posting Date:  October, 2012
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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

     
 
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Table of Contents

  • Introduction
  • 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
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: How to Set Up a FRAP Experiment

  Materials
  • cDNA of protein of interest (e.g., H‐Ras)
  • Fluorescent protein vector (e.g., EGFP)
  • Mammalian cultured cell lines (e.g., COS‐7, ATCC #CRL‐1651)
  • Cell culture medium (DMEM supplemented with 10% FBS and phenol red)
  • Transfection agent (e.g., Lipofectamine from Invitrogen)
  • Imaging medium (e.g., phenol red‐free DMEM supplemented with 10% FBS and 25 mM HEPES)
  • 35‐mm glass‐bottom dishes (e.g., MatTek, glass‐bottom microwell dishes)
  • Line‐scanning confocal microscope (e.g., Zeiss LSM 510 or comparable system), with the appropriate laser and filter configuration for the fluorophore to be used:
    • Argon ion laser (488 nm laser line for bleaching and for imaging EGFP and Alexa‐488)
    • HeNe lasers (only necessary if imaging red and far red fluorophore (i.e., Cy3, mCherry, Alexa‐555, Cy5, Alexa‐647, etc.)
    • Band‐pass emission filters (i.e., 505‐550 for EGFP and Alexa‐488)
    • Acquisition software (e.g., Zeiss LSM)
    • Objectives (e.g., 40×/1.3 Oil Plan‐Neofluar lens)
  • Stage and objective heater or imaging chamber
  • Software for image analysis (e.g., Zeiss LSM software, ImageJ)
  • Software for data analysis (e.g., Excel, MATLAB)
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Figures

Videos

Literature Cited

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