聚丙烯酰胺凝胶的蛋白质银染原理

聚丙烯酰胺凝胶旳蛋白质

银染原理主讲人：杨群峰2023-05-21Mechanismsofproteinsilverstaininginpolyacrylamidegels小构成员：杨群峰、李珍、唐棠、徐振杰、刘建云第1页1.Introduction措施长处缺陷考马斯亮蓝法银染法荧光染色负染色迅速简朴，反复性好，能在蒸馏水中保存数月条带也不会消失，成本较低，操作简便，无毒敏捷度最高(0.25ng)敏捷度较高(1-10ng),简朴迅速(30-60min)蛋白质保存完整，简朴迅速敏捷度相对较低，成果容易受到核酸旳污染，背景色较高背景较高，有毒，费时费力(8-16h),成本高，操作复杂使蛋白质变性，需要紫外透视仪反复性不好第2页1.IntroductionThesensitivitywasreportedtobeatleast100timesbetterthanforCoomassieBrilliantBlue.Silverstainingisacomplex,multistepprocess,andmanyvariablescaninfluencetheresult.High-purityreagentsandprecisetimingarenecessaryforreproducible,high-qualityresults.第3页2.Agstainingproceduresa.Fixing:

Treatwithacid,thisrendersthemacromoleculesinthegelinsolubleandpreventsthemfromdiffusingoutofthegelduringstainingsteps.Substancesinthegelthatinterferewithsilverstaining,suchasbuffers,ions,denaturants,detergents,orcarrierampholytes,arewashedoutofthegelduringthisstep.b.Sensitization:Treatwithreagents,thisrendersproteinsmorereactivetowardsilver,andreagentsthatacceleratethereductionofsilverion.第4页2.Agstainingproceduresc.Silverimpregnation:Treatwithsilvernitrate.Twofamiliesofmethodscanbeseparateddependingonthechemicalstateofthesilverionwhenusedforimpregatingthepolyacrylamidegel.(i)Thealkalinemethodsuseadiaminecomplexofsilvernitrateinahighlyalkalineenvironment(ammoniaandsodiumhydroxide).Development:diluteacidicsolutionsofformaldehyde.(ii)Theacidicmethodsusesilvernitrate(weaklyacidicsolution).Development:formaldehydesolutionsatalkalinepH.第5页2.Agstainingproceduresd.development:Ag++le→AgTheredoxpotentialofsilveris0.8eV.Twopropertiesofsilver:(i)Complexationdecreasestheredoxpotentialandthusthereducibilityofsilver.

(ii)Silvermetalstronglycatalyzesilverionreduction.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.第7页2.AgstainingproceduresItwillleadtopoorlysensitivenegativestainingwhenasilver-impregnatedgelisdippedintoaconventionalphotographicdeveloper.Method:insertawashbetweensilverimpregnationanddevelopment.第8页2.Agstainingprocedures1)Developmentwithalkalinemethods:Developmentisusuallyachievedbydiluteformaldehydesolutionscontainingsmallamountsofcitricacid.Thecitricacidconcentration:(i)lowconcentrations(highpH)ensurehighreductionspeedsandthushighsensitivitybutlowcontrastandhighbackground,(ii)highconcentrations(lowpH)slowdownthereductionspeedandensurelowbackgroundbutalsolowsensitivity.第9页2.AgstainingproceduresAsdevelopmentproceeds,silverionandalkalinecompoundsleakoutofthegel,slowingdownthereductionprocess.Thus,thesensitivityofthesemethodsislimitedifbackgroundisexpectedtobeclear,unlessenhancementstepsareincluded.2)Developmentwithacidicmethods:Washingmustbemuchshorter.Thereducingagent:formaldehydewithsmallamountsofalkali.第10页2.Agstainingprocedurese.Stoppingandgelpreservation:Thestoppingsolutionpreventsfurtherreductionofsilverion.Thepreservingsolutioncontainsglycerol,whichpreventsthegelfromcrackingduringdrying.EDTA:

Thiscompoundcomplexessilverionstrongly,preventingitsfurtherreductiontometallicsilverinthestoppingsolution.第11页3.Enhancementa.IncreasingsilverbindingCoattheproteinzoneswithSDS.b.DecreasingbackgroundimageTreatwithoxidants(e.g.dichromate)

priortosilverimpregnation.第12页3.Enhancementc.IncreasinglatentimageTreatwithsulfidingorreducingagents

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

Glutaraldehydereactscovalentlywithprotein,coveringitwithaldehydegroupsthatcanreactwithsilver,whichenhancesthesensitivityofsilverstaining.第13页4.

银染具体环节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%旳甲醛)显影至背景浮现7.定影：5%乙酸定影15min第14页4.

银染具体环节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直至背景浮现7.定影：1.5%EDTANa2定影10min。第15页5.影响敏捷度旳因素染色所选