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聚丙烯酰胺凝胶的蛋白质银染原理第1页1.Introduction方法优点缺点考马斯亮蓝法银染法荧光染色负染色快速简单，重复性好，能在蒸馏水中保留数月条带也不会消失，成本较低，操作简便，无毒灵敏度最高(0.25ng)灵敏度较高(1-10ng),简单快速(30-60min)蛋白质保留完整，简单快速灵敏度相对较低，结果轻易受到核酸污染，背景色较高背景较高，有毒，费时费劲(8-16h),成本高，操作复杂使蛋白质变性，需要紫外透视仪重复性不好聚丙烯酰胺凝胶的蛋白质银染原理第2页1.IntroductionThesensitivitywasreportedtobeatleast100timesbetterthanforCoomassieBrilliantBlue.Silverstainingisacomplex,multistepprocess,andmanyvariablescaninfluencetheresult.High-purityreagentsandprecisetimingarenecessaryforreproducible,high-qualityresults.聚丙烯酰胺凝胶的蛋白质银染原理第3页2.Agstainingproceduresa.Fixing:

Treatwithacid,thisrendersthemacromoleculesinthegelinsolubleandpreventsthemfromdiffusingoutofthegelduringstainingsteps.Substancesinthegelthatinterferewithsilverstaining,suchasbuffers,ions,denaturants,detergents,orcarrierampholytes,arewashedoutofthegelduringthisstep.b.Sensitization:Treatwithreagents,thisrendersproteinsmorereactivetowardsilver,andreagentsthatacceleratethereductionofsilverion.聚丙烯酰胺凝胶的蛋白质银染原理第4页2.Agstainingproceduresc.Silverimpregnation:Treatwithsilvernitrate.Twofamiliesofmethodscanbeseparateddependingonthechemicalstateofthesilverionwhenusedforimpregatingthepolyacrylamidegel.(i)Thealkalinemethodsuseadiaminecomplexofsilvernitrateinahighlyalkalineenvironment(ammoniaandsodiumhydroxide).Development:diluteacidicsolutionsofformaldehyde.(ii)Theacidicmethodsusesilvernitrate(weaklyacidicsolution).Development:formaldehydesolutionsatalkalinepH.聚丙烯酰胺凝胶的蛋白质银染原理第5页2.Agstainingproceduresd.development:Ag++le→AgTheredoxpotentialofsilveris0.8eV.Twopropertiesofsilver:(i)Complexationdecreasestheredoxpotentialandthusthereducibilityofsilver.

(ii)Silvermetalstronglycatalyzesilverionreduction.聚丙烯酰胺凝胶的蛋白质银染原理第6页2.AgstainingproceduresReductionspeed:mainlydependsonthefreesilverionconcentrationandontheprecisenatureofthereducingagent.Reductionspeed

isalsopH-dependent:RED+mOH-→OX+nH2O+me-e.g.HCHO+2OH-

→HCOOH+H2O+2e-Developmentisimmediatelystoppedwhenthegelisdippedintotheacidicsolution.Silverionscanbepresentinthreeforms:(i)free;(ii)complexedtothepolyacrylamidegel;(iii)complexedtoproteins,whichareknowntobindsilverquitestrongly.聚丙烯酰胺凝胶的蛋白质银染原理第7页2.AgstainingproceduresItwillleadtopoorlysensitivenegativestainingwhenasilver-impregnatedgelisdippedintoaconventionalphotographicdeveloper.Method:insertawashbetweensilverimpregnationanddevelopment.聚丙烯酰胺凝胶的蛋白质银染原理第8页2.Agstainingprocedures1)Developmentwithalkalinemethods:Developmentisusuallyachievedbydiluteformaldehydesolutionscontainingsmallamountsofcitricacid.Thecitricacidconcentration:(i)lowconcentrations(highpH)ensurehighreductionspeedsandthushighsensitivitybutlowcontrastandhighbackground,(ii)highconcentrations(lowpH)slowdownthereductionspeedandensurelowbackgroundbutalsolowsensitivity.聚丙烯酰胺凝胶的蛋白质银染原理第9页2.AgstainingproceduresAsdevelopmentproceeds,silverionandalkalinecompoundsleakoutofthegel,slowingdownthereductionprocess.Thus,thesensitivityofthesemethodsislimitedifbackgroundisexpectedtobeclear,unlessenhancementstepsareincluded.2)Developmentwithacidicmethods:Washingmustbemuchshorter.Thereducingagent:formaldehydewithsmallamountsofalkali.聚丙烯酰胺凝胶的蛋白质银染原理第10页2.Agstainingprocedurese.Stoppingandgelpreservation:Thestoppingsolutionpreventsfurtherreductionofsilverion.Thepreservingsolutioncontainsglycerol,whichpreventsthegelfromcrackingduringdrying.EDTA:

Thiscompoundcomplexessilverionstrongly,preventingitsfurtherreductiontometallicsilverinthestoppingsolution.聚丙烯酰胺凝胶的蛋白质银染原理第11页3.Enhancementa.IncreasingsilverbindingCoattheproteinzoneswithSDS.b.DecreasingbackgroundimageTreatwithoxidants(e.g.dichromate)

priortosilverimpregnation.聚丙烯酰胺凝胶的蛋白质银染原理第12页3.Enhancementc.IncreasinglatentimageTreatwithsulfidingorreducingagents

priortosilverimpregnation.Sulfidingagents:NH2CSNH2+H2O+2Ag+→NH2CONH2+Ag2S↓+2H+Reducingagents:

Glutaraldehydereactscovalentlywithprotein,coveringitwithaldehydegroupsthatcanreactwithsilver,whichenhancesthesensitivityofsilverstaining.聚丙烯酰胺凝胶的蛋白质银染原理第13页4.

银染详细步骤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%甲醛)显影至背景出现7.定影：5%乙酸定影15min聚丙烯酰胺凝胶的蛋白质银染原理第14页4.

银染详细步骤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3～5min直至背景出现7.定影：1.5%EDTANa2

定影10min。聚丙烯酰胺凝胶的蛋白质银染原理第15页5.影响灵敏度原因染色所选择温度、时间、银溶液含量、