Fluorography of polyacrylamide gels containing tritium.
Fluorography is the term used for the process of determining radioactivity in gels and other media by a combination of fluorescence and photography. Since most of the radiation of a low energy emitter will largely be absorbed by the gel, in the technique of fluorography a fluor (e.g., PPO) is infiltrated into the gel where it can absorb the radiation and re-emit light that will pass through the gel to the film. The resulting photographic image is analogous to an autoradiograph, but for a low energy beta-emitting isotope like (3)H, the sensitivity of fluorography is many times the sensitivity of autoradiography. The fluorograph may be used directly, as a qualitative picture of the radioactivity on the gel. It may also be used to locate radioactive bands or spots that can then be cut from the original gel for further analysis, or be scanned to give quantitative information about the distribution of radioactivity. Figure 1 shows an example of a gel that was stained with Coomassie blue and then fluorographed. Notice that there is no loss of resolution in the fluorography of thin gels of normal size. Fig. 1. This shows two examples of fluorography of protein bands labeled with (3)H. Basic nuclear proteins were isolated from the slime mold, Physarum polycephalum, pulse-labeled with (3)H-acetate in either S phase or G(2) phase of the naturally synchronous cell cycle. The proteins were analyzed by acrylamide gel electrophoresis in acetic acid, urea, and Triton X-100. After electrophoresis, the gel was stained with Coomassie blue, photographed, and then fluorographed. Individual lanes of the gel image were cut from the photograph (negative) and the fluorograph and then printed side-by-side to give the figure shown. Notice that the stain patterns of the two lanes are practically identical, except for the loading, while the radioactivity patterns show major differences, for example, the absence of label in his-tones H2A and H2B in G2 phase (4).
Waterborg
J H
JH
Department of Biological Chemistry, University of California School of Medicine, Davis, California.
Matthews
H R
HR