Detection and Quantitation of Radiolabeled Proteins in Gels and Blots

Daniel Voytas1, Ning Ke1

1 Iowa State University, Ames, Iowa
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 6.3
DOI:  10.1002/0471143030.cb0603s10
Online Posting Date:  May, 2001
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Abstract

Permanent records of the results of electrophoretic separations of radiolabeled proteins and membrane‐bound proteins (and RNA and DNA) can be made using autoradiography, fluorography, and phosphor imaging. These images can subsequently be quantified using densitometry to obtain a relative measure of the amount of radioactivity in a sample. This unit also includes descriptions of image‐enhancement techniques and guidelines for selection of appropriate recording media.

     
 
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Table of Contents

  • Basic Protocol 1: Autoradiography
  • Support Protocol 1: Fixing and Drying Gels for Autoradiography
  • Support Protocol 2: Use of Intensifying Screens
  • Support Protocol 3: Preflashing (Preexposing) Film
  • Alternate Protocol 1: Fluorography
  • Support Protocol 4: Densitometry
  • Alternate Protocol 2: Phosphor Imaging
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Autoradiography

  Materials
  • Fixed and dried gel (see protocol 2) or filter (e.g., from immunoblotting; unit 6.2)
  • Developer: Kodak developer and replenisher, prepared according to the manufacturer's instructions, 18° to 20°C
  • Fixer: Kodak fixer and replenisher, prepared according to the manufacturer's instructions, 18° to 20°C
  • Metal film cassette or paper film cassette with particle‐board supports and metal binder clips
  • Plastic wrap (e.g., Saran Wrap)
  • X‐ray film
  • Trays to hold film processing solutions
  • Clips for hanging film

Support Protocol 1: Fixing and Drying Gels for Autoradiography

  Materials
  • Gel from SDS‐PAGE (unit 6.1)
  • Fixing solution: 10% (v/v) glacial acetic acid/20% (v/v) methanol in H 2O
  • Alternative fixing solution (for gels with ≥15% acrylamide or thicker than 1.5 mm): 3% (v/v) glycerol/10% (v/v) glacial acetic acid/20% (v/v) methanol in H 2O
  • Glass dish
  • Rotary shaker
  • Filter paper (Whatman 3MM) in sheets at least 1 to 2 cm larger than gel
  • Plastic wrap (e.g., Saran Wrap)
  • Gel dryer with vacuum pump

Support Protocol 2: Use of Intensifying Screens

  Materials
  • Stroboscope or flash unit (e.g., Auto 22 Electronic Flash from Vivitar or Sensitize Pre‐Flash from Amersham Pharmacia Biotech)
  • Neutral‐density filter (Kodak)
  • Orange filter (Wratten 22; Kodak)
  • X‐ray film
  • Spectrophotometer

Support Protocol 3: Preflashing (Preexposing) Film

  Materials
  • Polyacrylamide gel
  • 1 M sodium salicylate, pH 5 to 7, freshly prepared
  • Additional reagents and equipment for fixing and drying gels (see protocol 2)

Alternate Protocol 1: Fluorography

  Materials
  • Gel or filter (e.g., from immunoblotting; unit 6.2)
  • PhosphorImager system (Molecular Dynamics) including:
  •  ImageEraser light box
  •  Exposure cassette with phosphor screen
  •  Scanning software
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Figures

Videos

Literature Cited

Literature Cited
   Chamberlain, J.P. 1979. Fluorographic detection of radioactivity in polyacrylamide gels with the water‐soluble fluor, sodium salicylate. Anal. Biochem. 98:132‐135.
   Johnston, R.F., Pickett, S.C., and Barker, D.L. 1990. Autoradiography using storage phosphor technology. Electrophoresis 11:355‐360.
   Laskey, R.A. 1980. The use of intensifying screens or organic scintillators for visualizing radioactive molecules resolved by gel electrophoresis. Methods Enzymol. 65:363‐371.
   Laskey, R.A. and Mills, A.D. 1975. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56:335‐341.
   Laskey, R.A. and Mills, A.D. 1977. Enhanced autoradiographic detection of 32P and 125I using intensifying screens and hypersensitized film. FEBS Lett. 82:314‐316.
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