Protein Protocols Protein Protocols

Autoradiography and Fluorography 307
38
Autoradiography and Fluorography of Acrylamide Gels
Antonella Circolo and Sunita Gulati
1. Introduction
Autoradiography detects the distribution of radioactivity on gels or filters by producing
permanent images on photographic film. It is frequently used in a variety of
experimental techniques ranging from Southern and Northern blot analysis (1), to visualization
of radioactive proteins separated in a sodium dodecyl sulfate (SDS)-Polyacrylamide
gels (2), to detection of nuclear factors bound to a labeled DNA probe in
gel shift analysis (3), and to localization of DNA bands in sequencing gels (4).
Autoradiographic images are formed when particles emitted by radioactive isotopes
encounter the emulsion of an X-ray film and cause emission of electrons from silver
halide crystals that, in turn, react with positively charged silver ions, resulting in the
precipitation of silver atoms and the formation of an image (5).
35S and 32P isotopes are the most commonly used isotopes for autoradiography. 35S is a β-emitter of relatively low energy (0.167 MeV). Thus 35S particles penetrate a film
to a depth of 0.22 mm, generally sufficient to interact with the emulsion in the film, as
long as care is taken to assure that the film and the source of radioactivity are in direct
contact, and that no barriers are posed between the film and the gel. In addition, gels
must be completely dry before autoradiography. 32P is a β-emitter with an energy of
1.71 MeV. Therefore, its particles penetrate water or other materials to a depth of
6 mm, passing completely through a film. In this case, gels or filters do not need to be
dry, since water will not block particles of this energy and may be covered with a clear
plastic wrap before autoradiography. The efficiency of 32P-emitted β-particles is
enhanced when an intensifying screen is placed behind the X-ray film, because radioactive
particles that pass through the film cause the screen to emit photons that sensitize
the film emulsion. The use of intensifying screens results in a fivefold increased
enhancement of the autoradiographic image when the exposure is performed at low
temperature (–70°C). In general, calcium tungstate screens are the most suitable
because they emit blue light to which X-ray films are very sensitive (6).
Radiation of sufficiently high energy (e.g., 32P and 35S ) can be detected by simple
autoradiography, but low energy emissions may not penetrate the coating of the film,
and the most sensitive fluorography procedure is used in these cases. In fluorography,
the use of fluorescent chemicals increases about 10-fold the sensitivity of detection of
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
307