Detection and Quantitation of Radiolabeled Proteins and DNA in Gels and Blots

Daniel Voytas1, Ning Ke1

1 Iowa State University, Ames, Iowa
Publication Name:  Current Protocols in Immunology
Unit Number:  Appendix 3J
DOI:  10.1002/0471142735.ima03js50
Online Posting Date:  November, 2002
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Abstract

This appendix presents procedures for visualizing and quantitating radiolabeled proteins or DNA separated by polyacrylamide gel electrophoresis or affixed to filter membranes. Autoradiography is the most common method by which this is accomplished; X‐ray film is the traditional recording medium. Film images can be analyzed by densitometry as described to obtain a quantitative measure of the radioactivity in the sample. The use of X‐ray films for autoradiography, however, suffers from lack of sensitivity and limited linear range. Protocols are provided for overcoming these limitations using fluorography and intensifying screens, both of which enhance the radioactive signal. Sensitivity and linear ranges of measurement can be greatly extended by using a phosphor imaging system, which also makes it much faster and easier to quantify radioactive samples.

     
 
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Table of Contents

  • Basic Protocol 1: Autoradiography
  • Support Protocol 1: Preparing 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
  • Dried gel (see protocol 2) or filter (e.g., from immunoblotting; unit 8.10)
  • 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 (optionally preflashed; see protocol 4)
  • Trays to hold film processing solutions
  • Clips for hanging film

Support Protocol 1: Preparing Gels for Autoradiography

  Materials
  • Polyacrylamide gel
  • Fixing solution: 10% glacial acetic acid/20% methanol (v/v) in water
  • Protein (for gels with ≥15% acrylamide or thicker than 1.5 mm: 3% glycerol/10% glacial acetic acid/20% methanol (v/v/v) in water
  • Glass dish
  • Rotary shaker
  • Filter paper (Whatman 3MM) in sheets at least 1 to 2 cm larger than gel
  • UV‐transparent 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 8.10)
  • 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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