聚丙烯酰胺凝胶的蛋白质银染原理.ppt

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 1 Introduction 1 Introduction Thesensitivitywasreportedtobeatleast100timesbetterthanforCoomassieBrilliantBlue Silverstainingisacomplex multistepprocess andmanyvariablescaninfluencetheresult High purityreagentsandprecisetimingarenecessaryforreproducible high qualityresults 2 Agstainingprocedures a Fixing Treatwithacid thisrendersthemacromoleculesinthegelinsolubleandpreventsthemfromdiffusingoutofthegelduringstainingsteps Substancesinthegelthatinterferewithsilverstaining suchasbuffers ions denaturants detergents orcarrierampholytes arewashedoutofthegelduringthisstep b Sensitization Treatwithreagents thisrendersproteinsmorereactivetowardsilver andreagentsthatacceleratethereductionofsilverion 2 Agstainingprocedures c Silverimpregnation Treatwithsilvernitrate Twofamiliesofmethodscanbeseparateddependingonthechemicalstateofthesilverionwhenusedforimpregatingthepolyacrylamidegel i Thealkalinemethodsuseadiaminecomplexofsilvernitrateinahighlyalkalineenvironment ammoniaandsodiumhydroxide Development diluteacidicsolutionsofformaldehyde ii Theacidicmethodsusesilvernitrate weaklyacidicsolution Development formaldehydesolutionsatalkalinepH 2 Agstainingprocedures d development Ag le AgTheredoxpotentialofsilveris0 8eV Twopropertiesofsilver i Complexationdecreasestheredoxpotentialandthusthereducibilityofsilver ii Silvermetalstronglycatalyzesilverionreduction 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 2 Agstainingprocedures Itwillleadtopoorlysensitivenegativestainingwhenasilver impregnatedgelisdippedintoaconventionalphotographicdeveloper Method insertawashbetweensilverimpregnationanddevelopment 2 Agstainingprocedures 1 Developmentwithalkalinemethods Developmentisusuallyachievedbydiluteformaldehydesolutionscontainingsmallamountsofcitricacid Thecitricacidconcentration i lowconcentrations highpH ensurehighreductionspeedsandthushighsensitivitybutlowcontrastandhighbackground ii highconcentrations lowpH slowdownthereductionspeedandensurelowbackgroundbutalsolowsensitivity 2 Agstainingprocedures Asdevelopmentproceeds silverionandalkalinecompoundsleakoutofthegel slowingdownthereductionprocess Thus thesensitivityofthesemethodsislimitedifbackgroundisexpectedtobeclear unlessenhancementstepsareincluded 2 Developmentwithacidicmethods Washingmustbemuchshorter Thereducingagent formaldehydewithsmallamountsofalkali 2 Agstainingprocedures e Stoppingandgelpreservation Thestoppingsolutionpreventsfurtherreductionofsilverion Thepreservingsolutioncontainsglycerol whichpreventsthegelfromcrackingduringdrying EDTA Thiscompoundcomplexessilverionstrongly preventingitsfurtherreductiontometallicsilverinthestoppingsolution 3 Enhancement a IncreasingsilverbindingCoattheproteinzoneswithSDS b DecreasingbackgroundimageTreatwithoxidants e g dichromate priortosilverimpregnation 3 Enhancement c IncreasinglatentimageTreatwithsulfidingorreducingagentspriortosilverimpregnation Sulfidingagents NH2CSNH2 H2O 2Ag NH2CONH2 Ag2S 2H Reducingagents Glutaraldehydereactscovalentlywithprotein coveringitwithaldehydegroupsthatcanreactwithsilver whichenhancesthesensitivityofsilverstaining 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 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3 5min直至背景出现7 定影 1 5 EDTANa2定影10min 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