Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells

Erik L. Snapp1, Nihal Altan1, Jennifer Lippincott‐Schwartz1

1 Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 21.1
DOI:  10.1002/0471143030.cb2101s19
Online Posting Date:  August, 2003
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Abstract

This unit describes fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) using commercially available confocal scanning laser microscopy (CSLM). Photobleaching is the photo‐induced change in a fluorphore that abolishes that molecule's fluorescence. The different characteristics of green fluorescent protein (GFP) chimeras in a cell can be studied by FRAP, in which a selected region of the cell is photobleached with intense light. The movement of unbleached molecules into a photobleached region is quantified by imaging with an attenuated light source. The movement of molecules between cellular compartments can be determined by FLIP, in which the same region of a cell expressing a GFP chimera is repeatedly photobleached. The loss of fluorescence from regions outside the photobleached region is monitored to characterize the movement of a protein. Together these two techniques are providing fundamentally new insights into the kinetic properties of proteins in cells.

     
 
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Table of Contents

  • Basic Protocol 1: Quantitative FRAP by Strip Photobleaching Using a Laser Scanning Confocal Microscope Capable of Selective Photobleaching
  • Alternate Protocol 1: FRAP with Confocal Laser Scanning Microscopes Without the Capacity for Selective Photobleaching
  • Support Protocol 1: Cell Transfection and Imaging Setup
  • Support Protocol 2: FRAP Data Processing
  • Alternate Protocol 2: Selective Photobleaching to Enhance Dim Structures while Imaging or to Visualize and Measure Nondiffusive Transport into an Organelle
  • Support Protocol 3: Simulation of Inhomogenous Diffusion
  • Basic Protocol 2: FLIP Using a Laser Scanning Confocal Microscope Capable of Selective Photobleaching
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Quantitative FRAP by Strip Photobleaching Using a Laser Scanning Confocal Microscope Capable of Selective Photobleaching

  Materials
  • Cell samples expressing GFP chimeric protein of interest ( protocol 3)
  • Confocal laser scanning microscope capable of selective photobleaching (e.g., Zeiss LSM 510)
  • Additional reagents and equipment for preparing imaging chambers and microscope for photobleaching experiments ( protocol 3)

Alternate Protocol 1: FRAP with Confocal Laser Scanning Microscopes Without the Capacity for Selective Photobleaching

  • Confocal laser scanning microscope without capacity for selective photobleaching (e.g., Zeiss LSM 410)

Support Protocol 1: Cell Transfection and Imaging Setup

  Materials
  • Eukaryotic cells of interest
  • Vector for expression of GFP chimeric protein: most laboratories today use the enhanced GFP (EGFP) variant (Clontech); although spectral variants of GFP are available, EGFP is best suited for photobleaching experiments; see Commentary for details
  • Cell culture medium (e.g., DMEM with serum) appropriate for cells of interest
  • Imaging medium (see recipe)
  • Silicon grease (optional)
  • 5 to 10 mg/ml poly‐L‐lysine (Sigma) in PBS (see appendix 2A for PBS)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Confocal laser scanning microscope equipped with a filter set for GFP and a 488‐nm excitation laser that is at least 10 mW and preferably ≥25 mW
  • Imaging chamber: chambered coverglass system (LabTek from Nalgene) or glass coverslips and silicon rubber gasket for constructing silicon rubber chamber (see recipe in unit 4.4; also see Fig. )
  • Immersion oil (for oil‐immersion microscope objectives)
  • Stage heater (e.g., Model ASI 400 Air Stream Stage Incubator, Nevtek; for cells that grow at 37°C)
  • Temperature probe: e.g., Thermolyne pyrometer (Cole‐Parmer)
  • Computer system capable of processing large image files: 350 MHz or faster processor, multiple gigabyte hard drive, and at least 128 MB RAM
  • Image processing software (i.e., NIH Image or Metamorph)
  • Additional reagents and equipment for transfection of eukaryotic cells by electroporation (unit 20.5) or use of lipid transfection reagents (unit 20.6)

Support Protocol 2: FRAP Data Processing

  Materials
  • Cell samples expressing GFP chimeric protein of interest ( protocol 3)
  • Confocal laser scanning microscope capable of selective photobleaching (e.g., Zeiss LSM 510)
  • Additional reagents and equipment for preparing imaging chambers and microscope for photobleaching experiments ( protocol 3)
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Figures

Videos

Literature Cited

Literature Cited
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