Ladner Carol L, Yang Jing, Turner Raymond J, Edwards Robert A
Structural Biology Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. N.W., Calgary, Alta., Canada, T2N 1N4.
Anal Biochem. 2004 Mar 1;326(1):13-20. doi: 10.1016/j.ab.2003.10.047.
2,2,2-Trichloroethanol (TCE) incorporated into polyacrylamide gels before polymerization provides fluorescent visible detection of proteins in less than 5min of total processing time. The tryptophans in proteins undergo an ultraviolet light-induced reaction with trihalocompounds to produce fluorescence in the visible range so that the protein bands can be visualized on a 300-nm transilluminator. In a previous study trichloroacetic acid or chloroform was used to stain polyacrylamide gel electrophoresis (PAGE) gels for protein visualization. This study shows that placing TCE in the gel before electrophoresis can eliminate the staining step. The gel is removed from the electrophoresis apparatus and placed on a transilluminator and then the protein bands develop their fluorescence in less than 5min. In addition to being rapid this visualization method provides detection of 0.2microg of typical globular proteins, which for some proteins is slightly more sensitive than the standard Coomassie brilliant blue (CBB) method. Integral membrane proteins, which do not stain well with CBB, are visualized well with the TCE in-gel method. After TCE in-gel visualization the same gel can then be CBB stained, allowing for complementary detection of proteins. In addition, visualization with TCE in the gel is compatible with two-dimensional PAGE, native PAGE, Western blotting, and autoradiography.
在聚合前将2,2,2-三氯乙醇（TCE）加入聚丙烯酰胺凝胶中，可在总处理时间不到5分钟的情况下对蛋白质进行荧光可见检测。蛋白质中的色氨酸与三卤化合物发生紫外线诱导反应，在可见光范围内产生荧光，从而使蛋白质条带能在300纳米的透射仪上显现出来。在先前的一项研究中，使用三氯乙酸或氯仿对聚丙烯酰胺凝胶电泳（PAGE）凝胶进行染色以观察蛋白质。本研究表明，在电泳前将TCE置于凝胶中可省去染色步骤。将凝胶从电泳装置中取出，放在透射仪上，然后蛋白质条带在不到5分钟内就会发出荧光。除了速度快之外，这种可视化方法可检测出0.2微克的典型球状蛋白质，对于某些蛋白质来说，其灵敏度略高于标准考马斯亮蓝（CBB）法。用CBB染色效果不佳的整合膜蛋白，用凝胶内TCE法能很好地显现出来。在凝胶内用TCE进行可视化后，同一块凝胶随后可用CBB染色，从而实现对蛋白质的互补检测。此外，凝胶内用TCE进行可视化与二维PAGE、天然PAGE、蛋白质印迹法和放射自显影兼容。
