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 Molecular Biology
Unit Number:  Appendix 3A
DOI:  10.1002/0471142727.mba03as48
Online Posting Date:  May, 2001
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Abstract

This unit presents procedures for visualizing and quantitating radiolabeled proteins or DNA separated by polyacrylamide gel electrophoresis (PAGE) or affixed to filter membranes. Procedures for autoradiography and densitometry are provided, but even greater sensitivity can be achieved with fluorography or by the use of intensifying screens, both of which enhance the radioactive signal. Preflashing the film is described to increase the linear measurement range for weakly radioactive samples. Sensitivity and linear ranges of measurement can also be greatly extended by using a phosphor imaging system to quickly and easily quantify radioactive samples.

     
 
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Table of Contents

  • Commonly Used Techniques in Biochemistry and Molecular Biology
  • 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 10.8)
  • 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 or 3% glycerol/10% glacial acetic acid/20% methanol (v/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 10.8)
  • 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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