2-D Electrophoresis.ppt

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1、2-DElectrophoresis二維電泳操作教學銘傳大學生物科技學系學生:賴柏融製96年11月前言此乃由操作二維電泳同學因需要而製作的教學簡報,僅供參考使用,不足的部分也請自行參照使用手冊或書籍,其中一定有不少遺漏或錯誤,敬請改正指教,此外要有教師指導後使用,避免因錯誤觀念而誤導.第1章　二維電泳(2-DElectrophoresis)簡介CoretechniqueofproteomicsCapableofsimultaneouslyresolvingthousandsofproteinsinoneseparationprocedureImmobili

2、zedpHgradientgels:higherresolutionimprovedinterlaboratoryreproducibilityhigherproteinloadingcapacityextendedbasicpHlimitSortsproteinsaccordingtotwoindependentpropertiesintwodiscretesteps:thefirst-dimensionstep,isoelectricfocusing(IEF),separatesproteinsaccordingtotheirisoelectricpi

3、nts(pI);thesecond-dimensionstep,SDS-polyacrylamidegelelectrophoresis(SDS-PAGE),separateproteinsaccordingtotheirmolecularweights.Eachspotontheresultingtwo-dimensionalarraycorrespondstoasingleproteinspeciesinthesampleThousandsofdifferentproteinscanthusbeseparated,andinformationsucha

4、stheproteinpI,theapparentmolecularweight,andtheamountofeachproteinisobtainedExperimentalsequencefor2-DelectrophoresisSamplepreparationIPGstriprehydrationIEFIPGstripequilibrationSDS-PAGEVisualizationAnalysis圖一Ettan™IPGphor™IsoelectricFocusingSystem第2章IEF儀器EttanIPGphorIsoelectricFoc

5、usingSystemCeramicmanifoldElectrodeassemblyRehydrationtrayPre-cutelectrodewicksSpiritlevelIPGCoverFluidCleaningsolutionCleaningbrush第3章　樣品的製備The2-DprocessbeginswithsamplepreparationAppropriatesamplepreparationisabsolutelyessentialforagood2-DresultKeepthesamplepreparationstrategyas

6、simpleaspossibletoavoidproteinlossesPreservesamplequalitybypreparingthesamplejustpriortoIEForstoringsamplesinaliquotsat-80°C第4章IPGStripRehydration(IPG膠條覆水作用）先將rehydrationtray(覆水槽)的蓋子打開，首先以ddH2O徹底沖洗覆水槽的凹槽，滴入數滴的EttanIPGphorStripHolderCleaningSolution(清潔液)，用Cleaningbrush(清潔刷子)仔細刷洗凹槽，

7、移除任何可能的殘留蛋白質，再用ddH2O沖洗乾淨後，徹底風乾。依照使用者手冊上的指示，配製適當體積的rehydrationsolution(覆水試劑)，依照兩組蛋白質樣本濃度的比例分別和lysisbuffer混合而成；接著加入適當體積的IPGBuffer。利用Spiritlevel(水平儀)來測量rehydrationtray的放置是水平的。將配好的rehydrationsolution分別注入要放置IPG膠條的凹槽。小心的將膠條上的保護塑膠條撕開，膠面朝下，輕輕的把膠條放入含有rehydrationsolution的凹槽，小心移動膠條，讓整條膠條都有覆蓋

8、住液體。小心不要造成小氣泡的產生。在凹槽中滴入IPGCoverFl