Rosenfeld J, Capdevielle J, Guillemot J C, Ferrara P
Laboratoire de Biochimie des Protéines, Sanofi Elf-BioRecherches, Labège Innopole, France.
Anal Biochem. 1992 May 15;203(1):173-9. doi: 10.1016/0003-2697(92)90061-b.
We examined the different steps necessary for the enzymatic digestion of proteins in the polyacrylamide matrix after gel electrophoresis. As a result, we developed an improved method for obtaining peptides for internal sequence analysis from 1-2 micrograms of in-gel-digested proteins. The long washing-lyophilization-equilibration steps necessary to eliminate the dye, sodium dodecyl sulfate, and other gel-associated contaminants that perturb protein digestion in Coomassie blue-stained gels have been replaced by washing for 40 min with 50% acetonitrile, drying for 10 min at room temperature, and then rehydrating with a protease solution. The washing and drying steps result in a substantial reduction of the gel slice volume that, when next swollen in the protease solution, readily absorbs the enzyme, facilitating digestion. The Coomassie blue staining procedure has also been modified by reducing acetic acid and methanol concentrations in the staining solution and by eliminating acetic acid in the destaining solution. The peptides resulting from the in-gel digestion are easily recovered by passive elution, in excellent yields for structural characterization. This simple and rapid method has been successfully applied for the internal sequence analysis of membrane proteins from the rat mitochondria resolved in preparative two-dimensional gel electrophoresis.
我们研究了凝胶电泳后在聚丙烯酰胺基质中进行蛋白质酶解所需的不同步骤。结果,我们开发了一种改进方法,可从1 - 2微克凝胶内消化的蛋白质中获取用于内部序列分析的肽段。在考马斯亮蓝染色凝胶中,消除干扰蛋白质消化的染料、十二烷基硫酸钠和其他与凝胶相关的污染物所需的长时间洗涤 - 冻干 - 平衡步骤,已被用50%乙腈洗涤40分钟、在室温下干燥10分钟,然后用蛋白酶溶液复水所取代。洗涤和干燥步骤使凝胶切片体积大幅减小,当凝胶切片随后在蛋白酶溶液中膨胀时,能很容易地吸收酶,从而促进消化。考马斯亮蓝染色程序也进行了修改,降低了染色溶液中的乙酸和甲醇浓度,并在脱色溶液中去除了乙酸。凝胶内消化产生的肽段通过被动洗脱很容易回收,用于结构表征时产率很高。这种简单快速的方法已成功应用于对在制备型二维凝胶电泳中分离的大鼠线粒体膜蛋白进行内部序列分析。