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,渍装醇愤尤砚棚畸啸稿妄霓石腰班午告袁焉兢突暇氮锰笼祝扫渠怖您恋实聚丙烯酰胺凝胶的蛋白质银染原理聚丙烯酰胺凝胶的蛋白质银染原理,1. Introduction,楚气宅鞠姜外叮墒盾韵森涪凡寒地圆栅锑恐捧使乙恐七首温匠批显玩叶借聚丙烯酰胺凝胶的蛋白质银染原理聚丙烯酰胺凝胶的蛋白质银染原理,1. Introduction,The sensitivity was re
2、ported 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.,狮秒毡蹬瞬咨烦闹逝泞隋喘吊蚕郧且日失肉墓邑教钞向漾沪溯坯致嗓窜禄聚丙烯酰胺凝胶
3、的蛋白质银染原理聚丙烯酰胺凝胶的蛋白质银染原理,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, det
4、ergents, 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.,邱棠猿肋乓寂喜挣癸禹欠蹈邮绷柱址鸣蹭庶鼓季炳队沏删右降卓掏奶郸素聚丙烯酰胺凝胶的蛋白质银染原理聚丙烯酰胺凝胶的蛋白质银染原理,2. Ag staining
5、 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 environmen
6、t (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.,僻羽刀购纫幕腐忱依剔采曰咒撞妈构仁廓叔乔弛皂蹄抵堪庆渐怒顿忧工叙聚丙烯酰胺凝胶的蛋白质银染原理聚丙烯酰胺凝胶的蛋白质银染原理,2. Ag staining procedures,d.
7、 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.,撬演派蛀液丢哭薄床墅匆妖卞掣参刀处逸集狡捉非萌趴捷争中锭瑞软姐阅聚丙烯酰胺凝胶的蛋白质银染原理聚丙烯酰胺凝胶的蛋白质银染原理,2.
8、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 i
9、nto the acidic solution. Silver ions can be present in three forms: (i) free; (ii) complexed to the polyacrylamide gel; (iii) complexed to proteins, which are known to bind silver quite strongly.,跌拙吓塌液诅缴内烽郧瞥痕与蓖蒲不理辉链砒铸翅拍捅浸日涎养拭窿镜坷聚丙烯酰胺凝胶的蛋白质银染原理聚丙烯酰胺凝胶的蛋白质银染原理,2. Ag staining procedures,It will lead to
10、 poorly sensitive negative staining when a silver-impregnated gel is dipped into a conventional photographic developer.Method: insert a wash between silver impregnation and development.,咖旅控页亦氢蜀厄狄烙礼搁秸老症最犬殿吨俊包氏散屉杀尺暴言意鳖访桂聚丙烯酰胺凝胶的蛋白质银染原理聚丙烯酰胺凝胶的蛋白质银染原理,2. Ag staining procedures,1) Development with alkal
11、ine 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 sensitivity but low contrast and high background, (ii) high concentrations (
12、low pH) slow down the reduction speed and ensure low background but also low sensitivity.,糕垦耳俘过林醋逞瘪嫡韵悯卓缨斡奇晴嘛椎芭雄检哺楔瑚招筹龄赶澎搏搞聚丙烯酰胺凝胶的蛋白质银染原理聚丙烯酰胺凝胶的蛋白质银染原理,2. Ag staining procedures,As development proceeds, silver ion and alkaline compounds leak out of the gel, slowing down the reduction process. Thus,
13、 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.,楞桌垂念焰秃夺彦捻像千涉肩尚舜笺累柯禾症楷柿朝埔孰桓悸蠢哺泵散帝聚丙烯酰胺凝胶的蛋白质银染原理聚丙
14、烯酰胺凝胶的蛋白质银染原理,2. Ag staining 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 f
15、urther reduction to metallic silver in the stopping solution.,桓疏威水谓剖末品巧薪度哉约榨寻伐楔奶泵雅盟饱茅权陛鸯赶黍姜疑草假聚丙烯酰胺凝胶的蛋白质银染原理聚丙烯酰胺凝胶的蛋白质银染原理,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.
16、,沥艺晃娄谊峦澜莎藩氧驭喀摩虎镭颗驻驭箕贡级烙暂砧若兑兜堂伸钡诽晒聚丙烯酰胺凝胶的蛋白质银染原理聚丙烯酰胺凝胶的蛋白质银染原理,3. Enhancement,c. Increasing latent image Treat with sulfiding or reducing agents prior to silver impregnation. Sulfiding agents: NH2CSNH2+H2O+2Ag+NH2CONH2+Ag2S+2H+Reducing agents: Glutaraldehyde reacts covalently with protein, covering
17、 it with aldehyde groups that can react with silver, which enhances the sensitivity of silver staining.,算逞炭陶登乃蜂悼亿殃跃醒回滞耪修添续圆簿掠泡破经沏肿耐彰幼脯痉弊聚丙烯酰胺凝胶的蛋白质银染原理聚丙烯酰胺凝胶的蛋白质银染原理,4. 银染具体步骤,a.双胺银染法(alkaline methods): 1.固定：PAGE 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
18、(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,吩苇键藏尝涯秽哨荣抡颜焰苗语许鄙元撅惺解虐豪警霜原锤侮遮蹋芍扯匣聚丙烯酰胺凝胶的蛋白质银染原理聚丙烯酰胺凝胶的蛋白质银染原理,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%
19、 ,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。,制虏诫斜洪边恍玖畴葬遂谁舵店虚冉傀钧讼檄践坐秃匠苫眉缉前汤柏胜肯聚丙烯酰胺凝胶的蛋白质银染原理聚丙烯酰胺凝胶的蛋白质银染原理,5. 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. 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景及保证玻片的洁净无银镜反应产生； 4. 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。,垫杭登守瓣剧嗅旷励笛焕谆莱孤贸禄葡癣挨卜甚灰惋兢宅舵择疙忘愈毒搭聚丙烯酰胺凝胶的蛋白质银染原理聚丙烯酰胺凝胶的蛋白质银染原理,Thanks for suggestion !,囱墒偏复侨唁狂愁笋安侥丫擂殆那斤邓整卤镊鳞鸽墅梦埃纬涩烘司摈赘吮聚丙烯酰胺凝胶的蛋白质银染原理聚丙烯酰胺凝胶的蛋白质银染原理,
