聚丙烯酰胺凝胶的蛋白质银染原理

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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2009-05-21Mechanismsofproteinsilverstaininginpolyacrylamidegels小组成员：杨群峰、李珍、唐棠、徐振杰、刘建云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，能在蒸馏水中保存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)蛋白质保存完整，简单快速灵敏度相对较低，结果容易受到核酸的污染，背景色较高背景较高，有毒，费时费力(8-16h),成

2、本高，操作复杂使蛋白质变性，需要紫外透视仪重复性不好1.IntroductionThesensitivitywasreportedtobeatleast100timesbetterthanforCoomassieBrilliantBlue.Silverstainingisacomplex,multistepprocess,andmanyvariablescaninfluencetheresult.High-purityreagentsandprecisetimingarenecessaryforreproducible,high-qualityresults.

3、2.Agstainingproceduresa.Fixing:Treatwithacid,thisrendersthemacromoleculesinthegelinsolubleandpreventsthemfromdiffusingoutofthegelduringstainingsteps.Substancesinthegelthatinterferewithsilverstaining,suchasbuffers,ions,denaturants,detergents,orcarrierampholytes,arewashedoutofthegeldu

4、ringthisstep.b.Sensitization:Treatwithreagents,thisrendersproteinsmorereactivetowardsilver,andreagentsthatacceleratethereductionofsilverion.2.Agstainingproceduresc.Silverimpregnation:Treatwithsilvernitrate.Twofamiliesofmethodscanbeseparateddependingonthechemicalstateofthesilverionwh

5、enusedforimpregatingthepolyacrylamidegel.(i)Thealkalinemethodsuseadiaminecomplexofsilvernitrateinahighlyalkalineenvironment(ammoniaandsodiumhydroxide).Development:diluteacidicsolutionsofformaldehyde.(ii)Theacidicmethodsusesilvernitrate(weaklyacidicsolution).Development:formaldehydes

6、olutionsatalkalinepH.2.Agstainingproceduresd.development:Ag++le→AgTheredoxpotentialofsilveris0.8eV.Twopropertiesofsilver:(i)Complexationdecreasestheredoxpotentialandthusthereducibilityofsilver.(ii)Silvermetalstronglycatalyzesilverionreduction.2.AgstainingproceduresReductionspeed:mai

7、nlydependsonthefreesilverionconcentrationandontheprecisenatureofthereducingagent.ReductionspeedisalsopH-dependent:RED+mOH-→OX+nH2O+me-e.g.HCHO+2OH-→HCOOH+H2O+2e-Developmentisimmediatelystoppedwhenthegelisdippedintotheacidicsolution.Silverionscanbepresentinthreeforms:(i)free;(ii)comp

8、lexedtothepolyacryl