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1、聚丙烯酰胺凝胶的蛋白质银染原理主讲人：杨群峰主讲人：杨群峰主讲人：杨群峰主讲人：杨群峰2009-05-212009-05-21Mechanisms of protein silver staining in polyacrylamide gels小组成员：杨群峰、李珍、唐棠、徐振杰、刘建云小组成员：杨群峰、李珍、唐棠、徐振杰、刘建云小组成员：杨群峰、李珍、唐棠、徐振杰、刘建云小组成员：杨群峰、李珍、唐棠、徐振杰、刘建云1. Introduction方法方法方法方法优点优点优点优点缺点缺点缺点缺点考马斯亮考马斯亮考马斯亮考马斯亮蓝法蓝法蓝法蓝法银染法银染法银染法银染法荧光染色荧光染色荧光染色荧光
2、染色负染色负染色负染色负染色快速简单，重复性好，能在快速简单，重复性好，能在快速简单，重复性好，能在快速简单，重复性好，能在蒸馏水中保存数月条带也不蒸馏水中保存数月条带也不蒸馏水中保存数月条带也不蒸馏水中保存数月条带也不会消失，成本较低，操作简会消失，成本较低，操作简会消失，成本较低，操作简会消失，成本较低，操作简便，无毒便，无毒便，无毒便，无毒灵敏度最高灵敏度最高灵敏度最高灵敏度最高(0.25ng)(0.25ng)灵敏度较高灵敏度较高灵敏度较高灵敏度较高(1-10ng),(1-10ng),简单快简单快简单快简单快速速速速(30-60min)(30-60min)蛋白质保存完整，简单快速蛋白质保
3、存完整，简单快速蛋白质保存完整，简单快速蛋白质保存完整，简单快速灵敏度相对较低，结果容易受到灵敏度相对较低，结果容易受到灵敏度相对较低，结果容易受到灵敏度相对较低，结果容易受到核酸的污染，背景色较高核酸的污染，背景色较高核酸的污染，背景色较高核酸的污染，背景色较高背景较高，有毒，费时费力背景较高，有毒，费时费力背景较高，有毒，费时费力背景较高，有毒，费时费力(8-(8-16h),16h),成本高，操作复杂成本高，操作复杂成本高，操作复杂成本高，操作复杂使蛋白质变性，需要紫外透视仪使蛋白质变性，需要紫外透视仪使蛋白质变性，需要紫外透视仪使蛋白质变性，需要紫外透视仪重复性不好重复性不好重复性不好重
4、复性不好1. Introduction The sensitivity was reported to be at least 100 times better than The sensitivity was reported to be at least 100 times better than for Coomassie Brilliant Blue.for Coomassie Brilliant Blue. Silver staining is a complex, multistep process, and many Silver staining is a complex, m
5、ultistep process, and many variables can influence the result. High-purity reagents and variables can influence the result. High-purity reagents and precise timing are necessary for reproducible, high-quality precise timing are necessary for reproducible, high-quality results.results.2. Ag staining
6、procedures a. Fixing:a. Fixing: Treat with acid, this renders the macromolecules in Treat with acid, this renders the macromolecules in the gel insoluble and prevents them from diffusing out of the the gel insoluble and prevents them from diffusing out of the gel during staining steps. Substances in
7、 the gel that interfere gel during staining steps. Substances in the gel that interfere with silver staining, such as buffers, ions, denaturants, with silver staining, such as buffers, ions, denaturants, detergents, or carrier ampholytes, are washed out of the gel detergents, or carrier ampholytes,
8、are washed out of the gel during this step.during this step. b. Sensitization: b. Sensitization: Treat with reagents, this renders proteins Treat with reagents, this renders proteins more reactive toward silver, and reagents that accelerate the more reactive toward silver, and reagents that accelera
9、te the reduction of silver ion.reduction of silver ion.2. Ag staining procedures c. Silver impregnation:c. Silver impregnation: Treat with silver nitrate. Treat with silver nitrate. Two families of methods can be separated depending on the Two families of methods can be separated depending on the ch
10、emical state of the silver ion when used for impregating the chemical state of the silver ion when used for impregating the polyacrylamide gel. polyacrylamide gel. (i) The alkaline methods use a diamine complex of silver (i) The alkaline methods use a diamine complex of silver nitrate in a highly al
11、kaline environment (ammonia and sodium nitrate in a highly alkaline environment (ammonia and sodium hydroxide). Development: dilute acidic solutions of hydroxide). Development: dilute acidic solutions of formaldehyde. formaldehyde. (ii) The acidic methods use silver nitrate (weakly acidic (ii) The a
12、cidic methods use silver nitrate (weakly acidic solution). Development: formaldehyde solutions at alkaline solution). Development: formaldehyde solutions at alkaline pH.pH.2. Ag staining procedures d. development:d. development: AgAg+ + + l e + l e AgAg The redox potential of silver is 0.8 eV.The re
13、dox potential of silver is 0.8 eV. Two properties of silver:Two properties of silver: (i) Complexation decreases the redox potential and thus the (i) Complexation decreases the redox potential and thus the reducibility of silver.reducibility of silver. (ii) Silver metal strongly catalyze silver ion
14、reduction.(ii) Silver metal strongly catalyze silver ion reduction.2. Ag staining procedures Reduction speed: mainly depends on the free silver ion Reduction speed: mainly depends on the free silver ion concentration and on the precise nature of the reducing agent.concentration and on the precise na
15、ture of the reducing agent. Reduction speedReduction speed is also pH-dependent:is also pH-dependent: RED + mOHRED + mOH- -OX + nHOX + nH2 2O + meO + me- - e. e.g. g. HCHO + 2 OHHCHO + 2 OH- - HCOOH + HHCOOH + H2 2O + 2eO + 2e- - Development is immediately stopped when the gel is dipped Development
16、is immediately stopped when the gel is dipped into the acidic solution. into the acidic solution. Silver ions can be present in three forms: (i) free; (ii) Silver ions can be present in three forms: (i) free; (ii) complexed to the polyacrylamide gel; (iii) complexed to complexed to the polyacrylamid
17、e gel; (iii) complexed to proteins, which are known to bind silver quite strongly.proteins, which are known to bind silver quite strongly.2. Ag staining procedures It will lead to poorly It will lead to poorly sensitive sensitive negativenegative staining when a silver-staining when a silver-impregn
18、ated gel is impregnated gel is dipped into a dipped into a conventional conventional photographic developer.photographic developer. Method:Method: insert a wash insert a wash between silver between silver impregnation and impregnation and development.development.2. Ag staining procedures 1) Developm
19、ent with alkaline methods:1) Development with alkaline methods: Development is usually achieved by dilute formaldehyde Development is usually achieved by dilute formaldehyde solutions containing small amounts of citric acid.solutions containing small amounts of citric acid. The citric acid concentra
20、tion:(i) low concentrations (high pH) The citric acid concentration:(i) low concentrations (high pH) ensure high reduction speeds and thus high sensitivity but low ensure high reduction speeds and thus high sensitivity but low contrast and high background, (ii) high concentrations (low contrast and
21、high background, (ii) high concentrations (low pH) slow down the reduction speed and ensure low pH) slow down the reduction speed and ensure low background but also low sensitivity.background but also low sensitivity.2. Ag staining procedures As development proceeds, silver ion and alkaline compound
22、s As development proceeds, silver ion and alkaline compounds leak out of the gel, slowing down the reduction process. leak out of the gel, slowing down the reduction process. Thus, the sensitivity of these methods is limited if background Thus, the sensitivity of these methods is limited if backgrou
23、nd is expected to be clear, unless enhancement steps are included.is expected to be clear, unless enhancement steps are included. 2) Development with acidic methods:2) Development with acidic methods: Washing must be much shorter. Washing must be much shorter. The reducing agent: formaldehyde with s
24、mall amounts of The reducing agent: formaldehyde with small amounts of alkali.alkali.2. Ag staining procedures e. Stopping and gel preservation: e. Stopping and gel preservation: The stopping solution The stopping solution prevents further reduction of silver ion. The preserving prevents further red
25、uction of silver ion. The preserving solution contains glycerol, which prevents the gel from solution contains glycerol, which prevents the gel from cracking during drying. cracking during drying. EDTA:EDTA: This compound complexes silver ion strongly, This compound complexes silver ion strongly, pr
26、eventing its further reduction to metallic silver in the preventing its further reduction to metallic silver in the stopping solution.stopping solution.3. Enhancement a. Increasing silver bindinga. Increasing silver binding Coat the protein zones with SDS.Coat the protein zones with SDS. b. Decreasi
27、ng background imageb. Decreasing background image Treat with oxidants (e.g. dichromate)Treat with oxidants (e.g. dichromate) prior to silver impregnation.prior to silver impregnation.3. Enhancement c. Increasing latent imagec. Increasing latent image Treat with sulfiding or reducing agentsTreat with
28、 sulfiding or reducing agents prior to silver prior to silver impregnation.impregnation. Sulfiding agents:Sulfiding agents: NHNH2 2CSNHCSNH2 2+H+H2 2O+2AgO+2Ag+ +NHNH2 2CONHCONH2 2+Ag+Ag2 2S S +2H+2H+ + Reducing agents:Reducing agents: Glutaraldehyde reacts covalently with protein, covering it with
29、Glutaraldehyde reacts covalently with protein, covering it with aldehyde groups that can react with silver, which enhances the aldehyde groups that can react with silver, which enhances the sensitivity of silver staining.sensitivity of silver staining.4. 银染具体步骤银染具体步骤a. a.双胺银染法双胺银染法双胺银染法双胺银染法( (alkal
30、ine methodsalkaline methods): ): 1. 1.固定固定固定固定：PAGE PAGE 胶在固定液胶在固定液(40%(40%乙醇乙醇,10%,10%乙酸乙酸) ) 中固定中固定1h 1h 2. 2.水洗：水洗：水洗：水洗：去离子水洗去离子水洗20min 220min 2次次 3. 3.敏化敏化敏化敏化：5%5%戊二醛浸泡戊二醛浸泡1h 1h 4. 4.水洗水洗水洗水洗：去离子水洗：去离子水洗40min 440min 4次次 5. 5.银染：银染：银染：银染：银氨液银氨液( (现用现配现用现配) )温育温育30min 30min 6. 6.显影：显影：显影：显影：显影液
31、显影液(50mg/L (50mg/L 柠檬酸柠檬酸,0.8ml 36%,0.8ml 36%的甲醛的甲醛) ) 显影显影至背景出现至背景出现 7. 7.定影：定影：定影：定影：5%5%乙酸定影乙酸定影15min15min4. 银染具体步骤银染具体步骤b.b.非双胺化学显色银染法非双胺化学显色银染法非双胺化学显色银染法非双胺化学显色银染法(acidic methods):(acidic methods): 1. 1.固定：固定：固定：固定：PAGE PAGE 胶在固定液胶在固定液(40%(40%乙醇乙醇,10%,10%乙酸乙酸) )中固定中固定30min30min 2. 2.敏化：敏化：敏化：敏化
32、：用致敏液用致敏液( (乙醇乙醇30% ,30% ,乙酸钠乙酸钠68% ,68% ,硫代硫酸钠硫代硫酸钠0.2% 0.2% , ,戊二醛戊二醛0.125%) 0.125%) 浸泡浸泡30min30min 3. 3.水洗：水洗：水洗：水洗：水洗水洗5min 35min 3次次 4. 4.银染：银染：银染：银染：银溶液银溶液( (硝酸银硝酸银0.25% ,0.25% ,甲醛甲醛0.015%) 0.015%) 作用作用30min ,30min , 5. 5.水洗：水洗：水洗：水洗：水洗水洗1min 21min 2次次 6. 6.显影：显影：显影：显影：显影液显影液( (碳酸钠碳酸钠2.5% ,2.5
33、% ,甲醛甲醛0.0074%) 0.0074%) 显影显影3 35min 5min 直至背景出现直至背景出现 7. 7.定影：定影：定影：定影：1.5%EDTANa1.5%EDTANa2 2 定影定影10min10min。5. 影响灵敏度的因素影响灵敏度的因素 染色所选择的温度、时间、银溶液的含量、洗涤的条件以染色所选择的温度、时间、银溶液的含量、洗涤的条件以及试剂对染色结果均有直接影响：及试剂对染色结果均有直接影响： 1. 1. 温度与时间：在不同的嗜银染色方法中均有不同的温度温度与时间：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；和染色时间要求； 2. 2. 溶液中银的含量对结果的
34、影响：在嗜银染色中银的含量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；是方法能否重复的关键； 3. 3. 水洗对染色结果的影响：在嗜银染色后还原前以去离子水洗对染色结果的影响：在嗜银染色后还原前以去离子水洗代替单纯吸除切片多余银溶液，可以减少切片黄色背水洗代替单纯吸除切片多余银溶液，可以减少切片黄色背景及保证玻片的洁净无银镜反应产生；景及保证玻片的洁净无银镜反应产生； 4. 4. 试剂的影响：乙醇、甲醛和戊二醛必须是新鲜试剂，无试剂的影响：乙醇、甲醛和戊二醛必须是新鲜试剂，无聚合或氧化，而且必须是分析纯试剂。聚合或氧化，而且必须是分析纯试剂。 Thanks for suggestion !
