聚丙烯酰胺凝胶的蛋白质银.pptVIP

主讲人：杨群峰 2009-05-21 Mechanisms of protein silver staining in polyacrylamide gels 小组成员：杨群峰、李珍、唐棠、徐振杰、刘建云 1. Introduction 方法 优点 缺点 考马斯亮蓝法 银染法 荧光染色 负染色 快速简单，重复性好，能在蒸馏水中保存数月条带也不会消失，成本较低，操作简便，无毒 灵敏度最高(0.25ng) 灵敏度较高(1-10ng),简单快速(30-60min) 蛋白质保存完整，简单快速 灵敏度相对较低，结果容易受到核酸的污染，背景色较高 背景较高，有毒，费时费力(8-16h),成本高，操作复杂 使蛋白质变性，需要紫外透视仪 重复性不好 1. Introduction The sensitivity was reported to be at least 100 times better than for Coomassie Brilliant Blue. Silver staining is a complex, multistep process, and many variables can influence the result. High-purity reagents and precise timing are necessary for reproducible, high-quality results. 2. Ag staining procedures a. Fixing: Treat with acid, this renders the macromolecules in the gel insoluble and prevents them from diffusing out of the gel during staining steps. Substances in the gel that interfere with silver staining, such as buffers, ions, denaturants, detergents, or carrier ampholytes, are washed out of the gel during this step. b. Sensitization: Treat with reagents, this renders proteins more reactive toward silver, and reagents that accelerate the reduction of silver ion. 2. Ag staining procedures c. Silver impregnation: Treat with silver nitrate. Two families of methods can be separated depending on the chemical state of the silver ion when used for impregating the polyacrylamide gel. (i) The alkaline methods use a diamine complex of silver nitrate in a highly alkaline environment (ammonia and sodium hydroxide). Development: dilute acidic solutions of formaldehyde. (ii) The acidic methods use silver nitrate (weakly acidic solution). Development: formaldehyde solutions at alkaline pH. 2. Ag staining procedures d. development: Ag+ + l e →Ag The redox potential of silver is 0.8 eV. Two properties of silver: (i) Complexation decreases the redox potential and thus the reducibility of silver. (ii) Silver metal strongly catalyze silver ion reduction. 2. Ag staining procedures Reduction speed: mainly de