聚丙烯酰胺凝胶的蛋白质银染原理

1、Mechanisms of protein silver staining in polyacrylamide gels2. Ag stainind, 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, deter

2、gents, 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 staininTreat with silver nitrate. Two families of methods can be separate

3、d 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 acidi

4、c methods use silver nitrate (weakly acidic solution). Development: formaldehyde solutions at alkaline pH.2. Ag staininAg 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 ca

5、talyze silver ion reduction.2. Ag stainin2. Ag stainin2. Ag staininaline 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 reduction speeds and thus high sensit

6、ivity but low contrast and high background, (ii) high concentrations (low pH) slow down the reduction speed and ensure low background but also low sensitivity.ds, silver ion and alkaline compounds leak out of the gel, slowing down the reduction process. Thus, the sensitivity of these methods is limi

7、ted 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 stainin3. Enhan3. Enhanage Treat with sulfiding or reducing agentsprior to silver im

8、pregnation. 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.银银染染hods): 1.固固定定：PAGE 胶在固定液(40%乙醇,10%乙酸) 中固定1h 2.水水洗：洗：去离

9、子水洗20min 2次 3.敏敏化化：5%戊二醛浸泡1h 4.水水洗洗：去离子水洗40min 4次 5.银银染：染：银氨液(现用现配)温育30min 6.显显影：影：显影液(50mg/L 柠檬酸,0.8ml 36%的甲醛) 显影至背景出现 7.定定影：影：5%乙酸定影15min4.银银染染ic methods): 1.固固定：定：PAGE 胶在固定液(40%乙醇,10%乙酸)中固定30min 2.敏敏化：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.水水洗：洗：水洗5min 3次 4.银银染：染：银溶液(硝酸银0.25% ,甲醛0.015%) 作用30min , 5.水水洗：洗：水洗1min 2次 6.显显影：影：显影液(碳酸钠2.5% ,甲醛0.0074%) 显影35min 直至背景出现 7.定定影：影：1.5%EDTANa2定影10min。5. 影影响响灵灵条件以及试剂对染色结果均有直接影响： 1. 温度与时间：在不同的嗜银染色方法中均有不同的温度和染色时间要求； 2. 溶液中银的含量对结果的影响：在嗜银染色中银的含量是方法能否重复的关键； 3.