聚丙烯酰胺凝胶的蛋白质银染原理

1、聚丙烯酰胺凝胶的蛋白质银染原理,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,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

2、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 sta

3、ining 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 accel

4、erate 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

5、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:

6、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 i

7、on 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. Silver ions can be present in three forms: (i) fre

8、e; (ii) complexed to the polyacrylamide gel; (iii) complexed to proteins, which are known to bind silver quite strongly.,2. Ag staining procedures,It will lead to poorly sensitive negative staining when a silver-impregnated gel is dipped into a conventional photographic developer. Method: insert a w

9、ash between silver impregnation and development.,2. Ag staining procedures,1) Development with alkaline methods: Development is usually achieved by dilute formaldehyde solutions containing small amounts of citric acid. The citric acid concentration:(i) low concentrations (high pH) ensure high reduct

10、ion speeds and thus high sensitivity but low contrast and high background, (ii) high concentrations (low pH) slow down the reduction speed and ensure low background but also low sensitivity.,2. Ag staining procedures,As development proceeds, silver ion and alkaline compounds leak out of the gel, slo

11、wing down the reduction process. Thus, the sensitivity of these methods is limited if background is expected to be clear, unless enhancement steps are included. 2) Development with acidic methods: Washing must be much shorter. The reducing agent: formaldehyde with small amounts of alkali.,2. Ag stai

12、ning procedures,e. Stopping and gel preservation: The stopping solution prevents further reduction of silver ion. The preserving solution contains glycerol, which prevents the gel from cracking during drying. EDTA: This compound complexes silver ion strongly, preventing its further reduction to meta

13、llic silver in the stopping solution.,3. Enhancement,a. Increasing silver binding Coat the protein zones with SDS. b. Decreasing background image Treat with oxidants (e.g. dichromate) prior to silver impregnation.,3. Enhancement,c. Increasing latent image Treat with sulfiding or reducing agents prio

14、r to silver impregnation. Sulfiding agents: NH2CSNH2+H2O+2Ag+NH2CONH2+Ag2S+2H+ Reducing agents: Glutaraldehyde reacts covalently with protein, covering it with aldehyde groups that can react with silver, which enhances the sensitivity of silver staining.,4. 银染具体步骤,a.双胺银染法(alkaline methods): 1.固定：PAG

15、E 胶在固定液(40%乙醇,10%乙酸) 中固定1h 2.水洗：去离子水洗20min 2次 3.敏化：5%戊二醛浸泡1h 4.水洗：去离子水洗40min 4次 5.银染：银氨液(现用现配)温育30min 6.显影：显影液(50mg/L 柠檬酸,0.8ml 36%的甲醛) 显影至背景出现 7.定影：5%乙酸定影15min,4. 银染具体步骤,b.非双胺化学显色银染法(acidic methods): 1.固定：PAGE 胶在固定液(40%乙醇,10%乙酸)中固定30min 2.敏化：用致敏液(乙醇30% ,乙酸钠68% ,硫代硫酸钠0.2% ,戊二醛0.125%) 浸泡30min 3.水洗：水洗5min 3次 4.银染：银溶液(硝酸银0.25% ,甲醛0.015%) 作用30min , 5.水洗：水洗1min 2次 6.显影：显影液(碳酸钠2.5% ,甲醛0.0074%) 显影35min 直至背景出现 7.定影：1.5%EDTANa2 定影10min。,5. 影响灵敏度的因素,染色所选择的温度、时间