Paech C, Christianson T, Maurer K H
COGNIS, Inc., Santa Rosa, California 95407.
Anal Biochem. 1993 Feb 1;208(2):249-54. doi: 10.1006/abio.1993.1041.
A simple, extremely versatile method for preparing zymograms from proteases after nondenaturing Phast-System gel electrophoresis is described. After completion of the run and before staining, an electropherogram and a piece of developed, single-side coated X-ray film are brought into contact for 5 min at room temperature. The film overlay is then separated and the gel is stained for protein. The zymogram on the X-ray film is generated by simply pouring about 10 ml of a suitable buffer solution at 30 to 50 degrees C over the film strip. Clearing zones appeared within a few seconds to a few minutes depending on protease amount. For the alkaline protease subtilisin BL the lower limit of detectability was 10 ng applied to the gel prior to electrophoresis. For preservation and archiving the zymogram film is simply rinsed with distilled water and air-dried. The film can be cut to size and mounted for a slide projector, or the film can serve as a negative for photographic enlargements. The clearing zone area is proportional to the amount of protease from 10 to 100 ng of protein.
本文描述了一种简单且用途极为广泛的方法，用于在非变性Phast-System凝胶电泳后从蛋白酶制备酶谱。电泳结束后且在染色前，将电泳图谱和一张显影后的单面涂布X射线胶片在室温下接触5分钟。然后分离胶片覆盖物，对凝胶进行蛋白质染色。只需在30至50摄氏度下将约10毫升合适的缓冲溶液倒在胶片条上，即可在X射线胶片上生成酶谱。根据蛋白酶量的不同，在几秒钟到几分钟内会出现清晰区。对于碱性蛋白酶枯草杆菌蛋白酶BL，电泳前施加到凝胶上的可检测下限为10纳克。为了保存和存档，酶谱胶片只需用蒸馏水冲洗并风干。胶片可以裁剪成合适尺寸并安装用于幻灯片投影仪，或者该胶片可以用作照相放大的底片。清晰区面积与10至100纳克蛋白质的蛋白酶量成正比。
