Waterborg J H, Matthews H R
Department of Biological Chemistry, University of California School of Medicine, Davis, California.
Methods Mol Biol. 1984;1:147-52. doi: 10.1385/0-89603-062-8:147.
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).
荧光自显影术是一个术语，用于通过荧光和摄影相结合的方法来测定凝胶及其他介质中的放射性。由于低能发射体发出的大部分辐射会被凝胶大量吸收，在荧光自显影技术中，一种荧光物质（如PPO）会渗入凝胶，在那里它可以吸收辐射并重新发射出能穿过凝胶到达胶片的光。所得的照片图像类似于放射自显影片，但对于像³H这样的低能β发射同位素，荧光自显影的灵敏度是放射自显影灵敏度的许多倍。荧光自显影片可直接用作凝胶上放射性的定性图像。它也可用于定位放射性条带或斑点，然后从原始凝胶上切下进行进一步分析，或者进行扫描以给出有关放射性分布的定量信息。图1展示了一个用考马斯亮蓝染色后再进行荧光自显影的凝胶示例。注意，正常尺寸的薄凝胶进行荧光自显影时分辨率没有损失。图1. 这展示了用³H标记的蛋白质条带的荧光自显影的两个示例。从多头绒泡菌中分离出碱性核蛋白，在自然同步细胞周期的S期或G₂期用³H - 乙酸盐进行脉冲标记。通过在乙酸、尿素和 Triton X - 100 中进行丙烯酰胺凝胶电泳对蛋白质进行分析。电泳后，凝胶用考马斯亮蓝染色、拍照，然后进行荧光自显影。凝胶图像的各个泳道从照片（底片）和荧光自显影片上剪下，然后并排打印以得到所示的图。注意，除了加样情况外，两条泳道的染色模式几乎相同，而放射性模式显示出主要差异，例如，G₂期组蛋白H2A和H2B中没有标记（4）。
