双向电泳1D技术相关.ppt

2DElectrophoresis 1stDimension ProteomicsExperimentalWorkflow Isoelectricfocusing IPGrehydration IPGsaresuppliedasdrystrips onaflexibleplasticsupportTheymustberehydratedtoatleasttheiroriginalvolume 0 5mmthickx3 3mmwide Rehydrationcanbedoneinafocusingtrayoradisposablerehydrationtray Focusingtray Rehydrationtray Firstdimension whatistheisoelectricpoint pI ProteinsareamphotericThechargesaredeterminedbyaminoacidcompositionpHoftheenvironmentIsoelectricpointIEFiselectrophoresisofproteinsinapHgradient ChargedproteinsmoveinanelectricfielduntiltheyreachthepointinthepHgradientwheretheirnetchargeisneutral ProteinsmoveinapHgradientuntiltheyreachtheirisoelectricpoint pI Focusingwithvoltage 9 3 6 6 3 5 4 3 10 8 7 8 4 7 5 9 4 7 6 5 3 4 9 10 5 10 8 6 9 3 3 3 3 4 4 4 4 5 5 5 5 6 6 6 6 6 7 7 7 8 8 8 9 9 9 9 10 10 10 Cathode Anode Anode Cathode pH pH Firstdimension whatisisoelectricfocusing IEF HowdoyourunIEF SeparationMedium IPGStripsInstrument IEFCell IPGStrips IPGstandsforimmobilizedpHgradientAcrylamidegelsarepouredwithapHgradientontoaplasticbacking cutintostripsanddehydratedpHgradientsarecreatedwithsetsofacrylamidobufferswhicharederivativesofacrylamidecontainingbothreactivedoublebondsandbufferinggroupsThepHgradientisfixedinthegelanddoesn tchangeduringfocusingIPGstripsallowforahighdegreeofreproducibilityandeasyhandling IPGstrips IPGstripsareavailableinavarietyofpHgradientsandlengths ReadyStrip IPGstrips 7cm11cm17cm18cm24cm Lengths BroadrangepH3 10pH3 10nonlinear NL NarrowRangepH3 6pH4 7pH5 8pH7 10MicroRangepH3 9 5 1pH4 7 5 9pH5 5 6 7pH6 3 8 3 BroadRangepISeparations pH3 10 pH3 10NL IPGStripSelection pH3 10 pH3 6 pH5 8 pH7 10 E coliLysate IPGStripResolution Narrow Micro Broad 3 10 5 8 4 7 5 9 Sample RehydrationbufferforIEF ReadyPrepRehydration SampleBuffer8Murea 2 CHAPS 50mMdithiothreitol DTT 0 2 w v Bio Lyte 3 10ampholytes BromophenolBlue问题 1加入这些目的是什么 2可以有替代品吗 为什么不加其他东西 PCR反应体系 LaemmliSampleBufferfor1DSDS PAGE WhydoyouuseSDSin1DSDS PAGEbufferandnotinrehydration samplebuffer Whydoyouuseglycerolin1DSDS PAGEbuffer WhydoyouuseDTTinboth1DSDS PAGEbufferandrehydrationsamplebuffer ProteinSolubility 加了一大堆东西 那么在什么情况下蛋白最容易溶解 Separatepolypeptidechains如何能够达到这个目的呢 BreakingMolecularInteractions这些相互作用都包括什么呢 covalentinteractions disulfidebridgesnon covalentinteractions ionicbonds hydrogenbondshydrophobicinteractions DisruptionofDisulfideBridges Reducingagent1Mercaptoethanol 700mM碱性端的离子化 会破坏pH梯度2Dithiothreitol DTT 50mM无离子化 但并非完美 较多的半胱氨酸二硫键难以被还原3Tributylphosphine TBP 2mM挥发性 毒性 难闻气味 需要有机溶剂溶解 没有文献报道效果比DTT好 DisruptionofIonicBond HydrogenBond Chaotrope改变介电常数 氢键形成和极化 疏水键 几乎所有和蛋白质溶解相关的参数 1Urea最主要的功能是打破氢键 其次是离子键 而打破离子键是通过改变节电常数和蛋白质变性 2Thiourea很强的致蛋白质变性作用 与Urea联合使用 明显促进蛋白质溶解 问题 可以单独使用吗 DisruptionofHydrophobicInteractions Detergent 1Ionic SDS完全不能用吗 2Nonionic Triton tween NP 40 Brij Mega能用吗 3Zwitterionic CHAPS ASB能用吗 由于Urea的原因 在高浓度Urea情况下 CHAPS Triton NP 40可以使用 Sample RehydrationbufferforIEF Isoelectricfocusing rehydration sampleloading PassiveRehydration novoltage sampleisinrehydrationbuffer ActiveRehydration lowvoltage sampleisinrehydrationbuffer CupLoading rehydratewithbuffer addsampletocupwithlowvoltage Prosandconsofdifferentrehydrationtechniques Powerconditions programming StripsshouldalwaysberunatthehighestvoltagecompatiblewiththeheatdissipationcapacitiesofthecellVolthours timeintegralofappliedvoltage Astandardforreproducingfocusingconditions ThisneedstobedeterminedempiricallyWhenanelectricalfieldisappliedtoanIPGstripatthebeginningofarun thecurrentwillbehighbecauseofthehighnumberofchargedcarrierspresent astheproteinsandampholytesmigrate thecurrentwillgraduallydecreasebecauseofdecreasingchargeSimilarstripsandsamplesshoul