Measuring Dynamics of Nuclear Proteins by Photobleaching

Miroslav Dundr1, Tom Misteli1

1 National Cancer Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 13.5
DOI:  10.1002/0471143030.cb1305s18
Online Posting Date:  May, 2003
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Abstract

The mobility of nuclear proteins can be studied by photobleaching techniques. The three main advantages of photobleaching are fast experimental turn around, good spatial and temporal resolution, and the ability to measure kinetics inside of living cells. The main disadvantage of these techniques is the requirement for fluorescently tagged proteins that have rigorously tested to ensure it has the same properties and function as its native counterpart. Three major methods of photobleaching microscopy are described: fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and inverse fluorescence recovery after photobleaching (iFRAP). Each of these techniques has characteristics permitting the determination of distinct parameters of protein behavior in vivo.

     
 
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Table of Contents

  • Basic Protocol 1: Fluorescence Recovery After Photobleaching (FRAP)
  • Alternate Protocol 1: Fluorescence Loss in Photobleaching (FLIP)
  • Alternate Protocol 2: Inverse Fluorescence Recovery After Photobleaching (iFRAP)
  • Support Protocol 1: Transient Transfection of Mammalian Cells by Electroporation
  • Support Protocol 2: Transient Transfection of Mammalian Cells Using FuGENE 6
  • Support Protocol 3: Quantification of Fluoresccently Tagged Protein Molecules in Nuclear Compartments of Single Living Cells
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Fluorescence Recovery After Photobleaching (FRAP)

  Materials
  • 2‐ or 4‐chambered Lab‐Tek II coverglass (Nalgene Nunc) containing cells, 50% to 70% confluent
  • Complete normal growth medium with 25 mM HEPES, but without phenol red (unit 1.2)
  • Confocal microscope
  • Air stream incubator (Nevtek)

Alternate Protocol 1: Fluorescence Loss in Photobleaching (FLIP)

  Materials
  • Cells
  • Complete growth medium (e.g., unit 1.2)
  • Plasmid DNA
  • Carrier DNA: sheared salmon sperm DNA
  • 1× PBS ( appendix 2A)
  • 0.25% (w/v) trypsin/1 mM EDTA, 37°C
  • Complete growth medium with 25 mM HEPES, but without phenol red
  • 10‐cm Petri dish
  • 37°C warming tray
  • 15‐ml test tubes
  • Electroporation cuvettes with 2‐mm gap
  • BTX ECM830 electroporator (BTX, a division of Genetronics, Inc.) or equivalent
  • Culture dish or incubation chamber (e.g., 2‐ or 4‐chambered Lab‐Tek II coverglass; Nalgene Nunc)
Additional reagents and equipment for mammalian cell culture (unit 1.1)NOTE: All solutions and materials coming into contact with cells must be sterile, and proper aseptic technique must be used.NOTE: All cell culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise indicated.NOTE: Avoid repeated freezing and defrosting of plasmid DNA stock solution. It may cause nicks in the plasmid DNA which leads to linearization of supercoiled DNA. Prepare aliquots of plasmid DNA and store them at −20°C.

Alternate Protocol 2: Inverse Fluorescence Recovery After Photobleaching (iFRAP)

  Materials
  • Cells
  • Serum‐containing and serum‐free medium (unit 1.2)
  • FuGENE 6 reagent (Roche)
  • Plasmid DNA solution
  • Serum‐containing medium (unit 1.2)
  • Serum (optional)
  • Glass coverslips or Lab Tek II incubation chambers (Nalgene Nunc)
  • Additional reagents and equipment for mammalian cell culture (unit 1.1)
NOTE: All solutions and materials coming into contact with cells must be sterile, and proper aseptic technique must be used.NOTE: All cell culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise indicated.NOTE: Avoid repeated freezing and defrosting of plasmid DNA stock solution. It may cause nicks in the plasmid DNA which leads to linearization of supercoiled DNA. Prepare aliquots of plasmid DNA and store them at −20°C.

Support Protocol 1: Transient Transfection of Mammalian Cells by Electroporation

  Materials
  • Cells expressing GFP‐tagged protein of interest (see protocol 4 and protocol 5)
  • Complete growth medium (unit 1.2)
  • 1× PBS ( appendix 2A)
  • GFP‐labeled VLP particles (Charpilienne et al., ; Dundr et al., )
  • 22 × 22–mm glass coverslips
  • Confocal microscope with 100× objective
  • Additional reagents and materials for mammalian cell culture (unit 1.1)
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Figures

Videos

Literature Cited

Literature Cited
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