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,主讲人：杨群峰2009-05-21,Mechanismsofproteinsilverstaininginpolyacrylamidegels,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,Thesensitivitywasreportedtobeatleast100timesbetterthanforCoomassieBrilliantBlue.Silverstainingisacomplex,multistepprocess,andmanyvariablescaninfluencetheresult.High-purityreagentsandprecisetimingarenecessaryforreproducible,high-qualityresults.,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的蛋白质银染原理聚丙烯酰胺凝胶的蛋白质银染原理,2.Agstainingprocedures,a.Fixing:Treatwithacid,thisrendersthemacromoleculesinthegelinsolubleandpreventsthemfromdiffusingoutofthegelduringstainingsteps.Substancesinthegelthatinterferewithsilverstaining,suchasbuffers,ions,denaturants,detergents,orcarrierampholytes,arewashedoutofthegelduringthisstep.b.Sensitization:Treatwithreagents,thisrendersproteinsmorereactivetowardsilver,andreagentsthatacceleratethereductionofsilverion.,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.Agstainingprocedures,c.Silverimpregnation:Treatwithsilvernitrate.Twofamiliesofmethodscanbeseparateddependingonthechemicalstateofthesilverionwhenusedforimpregatingthepolyacrylamidegel.(i)Thealkalinemethodsuseadiaminecomplexofsilvernitrateinahighlyalkalineenvironment(ammoniaandsodiumhydroxide).Development:diluteacidicsolutionsofformaldehyde.(ii)Theacidicmethodsusesilvernitrate(weaklyacidicsolution).Development:formaldehydesolutionsatalkalinepH.,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.Agstainingprocedures,d.development:Ag+leAgTheredoxpotentialofsilveris0.8eV.Twopropertiesofsilver:(i)Complexationdecreasestheredoxpotentialandthusthereducibilityofsilver.(ii)Silvermetalstronglycatalyzesilverionreduction.,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.Agstainingprocedures,Reductionspeed:mainlydependsonthefreesilverionconcentrationandontheprecisenatureofthereducingagent.ReductionspeedisalsopH-dependent:RED+mOH-OX+nH2O+me-e.g.HCHO+2OH-HCOOH+H2O+2e-Developmentisimmediatelystoppedwhenthegelisdippedintotheacidicsolution.Silverionscanbepresentinthreeforms:(i)free;(ii)complexedtothepolyacrylamidegel;(iii)complexedtoproteins,whichareknowntobindsilverquitestrongly.,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.Agstainingprocedures,Itwillleadtopoorlysensitivenegativestainingwhenasilver-impregnatedgelisdippedintoaconventionalphotographicdeveloper.Method:insertawashbetweensilverimpregnationanddevelopment.,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.Agstainingprocedures,1)Developmentwithalkalinemethods:Developmentisusuallyachievedbydiluteformaldehydesolutionscontainingsmallamountsofcitricacid.Thecitricacidconcentration:(i)lowconcentrations(highpH)ensurehighreductionspeedsandthushighsensitivitybutlowcontrastandhighbackground,(ii)highconcentrations(lowpH)slowdownthereductionspeedandensurelowbackgroundbutalsolowsensitivity.,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.Agstainingprocedures,Asdevelopmentproceeds,silverionandalkalinecompoundsleakoutofthegel,slowingdownthereductionprocess.Thus,thesensitivityofthesemethodsislimitedifbackgroundisexpectedtobeclear,unlessenhancementstepsareincluded.2)Developmentwithacidicmethods:Washingmustbemuchshorter.Thereducingagent:formaldehydewithsmallamountsofalkali.,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烯酰胺凝胶的蛋白质银染原理,2.Agstainingprocedures,e.Stoppingandgelpreservation:Thestoppingsolutionpreventsfurtherreductionofsilverion.Thepreservingsolutioncontainsglycerol,whichpreventsthegelfromcrackingduringdrying.EDTA:Thiscompoundcomplexessilverionstrongly,preventingitsfurtherreductiontometallicsilverinthestoppingsolution.,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.IncreasingsilverbindingCoattheproteinzoneswithSDS.b.DecreasingbackgroundimageTreatwithoxidants(e.g.dichromate)priortosilverimpregnation.,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.IncreasinglatentimageTreatwithsulfidingorreducingagentspriortosilverimpregnation.Sulfidingagents:NH2CSNH2+H2O+2Ag+NH2CONH2+Ag2S+2H+Reducingagents:Glutaraldehydereactscovalentlywithprotein,coveringitwithaldehydegroupsthatcanreactwithsilver,whichenhancesthesensitivityofsilverstaining.,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(alkalinemethods):1.固定：PAGE胶在固定液(40%乙醇,10%乙酸)中固定1h2.水洗：去离子水洗20min2次3.敏化：5%戊二醛浸泡1h4.水洗：去离子水洗40min4次5.银染：银氨液(现用现配)温育30min6.显影：显影液(50mg/L柠檬酸,0.8ml36%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(acidicmethods):1.固定：PAGE胶在固定液(40%乙醇,10%乙酸)中固定30min2.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30min3.水洗：水洗5min3次4.银染：银溶液(硝酸银0.25%,甲醛0.015%)作用30min,5.水洗：水洗1min2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