聚丙烯酰胺凝胶的蛋白质银染原理.pptVIP

聚丙烯酰胺凝胶的蛋白质银染原理 主讲人：杨群峰 2009-05-21 1. Introduction 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 depends on the free silver ion concentration and on the precise nature of the reducing agent. Reduction speed is also pH-dependent: RED + mOH-→OX + nH2O + me- e.g. HCHO + 2 OH- → HCOOH + H2O + 2e- Development is immediately stopped when the gel is dipped into the acidic solution. Silve