Chapter9

ElectrophoresisElectrophoresis區(qū)帶電泳載體電泳無載體電泳自由電泳毛細(xì)管電泳粉末電泳聚焦電泳紙電泳凝膠電泳電泳色譜淀粉電泳瓊脂糖凝膠電泳聚丙烯酰胺凝膠電泳等電聚焦電泳密度梯度電泳親和電泳雙向電泳

PAGE（聚丙烯酰胺凝膠電泳分類）NativePAGESDSPAGE變性還原電泳+βME

變性非還原電泳W(wǎng)ithoutβME陽極電泳陰極電泳連續(xù)電泳非連續(xù)電泳連續(xù)電泳非連續(xù)電泳8.1electrophoresisPrinciplesofelectrophoresiselectrophoresismethodsNative

polyacrylamidegelelectrophoresis(PAGE)Sodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS)Isoelectricfocusingelectrophoresis(IEF)2D-electrophoresisCapillaryelectrophoresis(CE)毛細(xì)管電泳Free－flowelectrophoresis(FFE)自由流電泳PrinciplesofelectrophoresisElectrophoresismaybedefinedasthemigrationofchargedionsinanelectricfield.MigrationvelocityMolecularweightMolecularsizepI■影響電泳泳動率的因素：外加電流電壓分子量分子形狀分子的等電點(diǎn)VoltageCATHODE陰極ANODE陽極+--+JuangRH(2004)ECXTheeffectofbufferpH

influencethecharge(q)ontheproteinandhencethedirectionandspeedofitsmigration.ionicstrength離子強(qiáng)度

influencestheproportionofthecurrentcarriedbytheproteinsatlowionicstrengththeproteinswillcarryarelativelylargeproportionofthecurrentandsowillhavearelativelyfastmigration.(0.02~0.2)PAGE/SDSSDSSDSAssemblingtheglassplatePouringthegelsseparatinggel分離膠;stackinggel濃縮膠

PreparingthesampleMixtheproteinsolutionwiththesamplebuffer(containingβ-mercaptoethanol巰基乙醇

andSDS)1.4

gramsofSDSbindspergramofproteinnegativelycharged荷負(fù)電的

protein–SDScomplexeshavethesamechargeperunitlengthMercaptoethanolassiststheproteindenaturationbyreducingalldisulfidebonds.HeatLoadingthesamplesRunningthegelRemovingandstainingthegel(usuallyCoomassiebrilliantblue考馬斯亮藍(lán))Acrylamidestock&AcrylamidegelSamplePreparationBuffer&ElectrophoresisHowlongtorunthegel?SDS■三種不同性質(zhì)蛋白質(zhì)的電泳比較：Molecular

WeightpINativePAGESDS-PAGEMobilityProteinQuaternary

StructureXYZTetramer(40,000)x4Monomer88,000Monomer60,0005.85.29.3FastSlowMediumSlowUpwardFastXYZ--+四級結(jié)構(gòu)分子量四聚體單體XYZ+SDSNativeSDS分子量及凈電荷密度均影響電泳速率只有分子量影響電泳速率XYZ-+-IsoelectricfocusingProteinsareampholytes（兩性電解質(zhì)）positivecharge(pH<PI)negativecharge(pH>PI)proteinsaredistributedthroughoutasolutioninapHgradient.theanodeatthelowpHendMoleculesinthelowpHzone(whichwillbepositivelycharged)willmigratetothecathode陽極.Theywillgraduallylosetheirpositivechargeandtheirrateofmigration遷移率willslowdown.IsoelectricfocusingWheneachproteinreachesapositionwherethepHisequaltoitspI,itwillloseallofitschargeanditsmigrationwillcease.Afterasufficienttime,therefore,therespectivemoleculeswillinconsequencebecome“focused”attheirisoelectricpoints.Disadvantages++■等電聚焦9753-0Ampholyte+聚焦在等電點(diǎn)在低pH處帶正電被排斥-+在高pH處帶負(fù)電被排斥--JuangRH(2004)ECXPreparativeIEF柱式：由兩性電解質(zhì)、樣品和蔗糖等制備的重液，與不含蔗糖的兩性電解質(zhì)和樣品組成輕液，通過梯度混合器加到電泳柱中，形成下重上輕的密度梯度。電泳需冷卻系統(tǒng)。電泳過程中，電流逐漸下降，到穩(wěn)定時，電泳結(jié)束。關(guān)掉電源，從柱下端小心緩慢地放出電泳液，根據(jù)需要收集流出液。旋管等電聚焦電泳：將樣品與兩性電解質(zhì)混合，裝入一個長的管內(nèi)，將其繞在圓柱上。將管兩端放到電極槽中，通電進(jìn)行電泳。結(jié)束后，直接將螺旋管分割成段，倒出電泳液，即可達(dá)到分離目的。制備量小。水平旋轉(zhuǎn)等電聚焦電泳：電泳柱橫放，柱內(nèi)多個平行多孔的聚酯膜將其分隔多個間隔部分。電泳柱繞中心軸旋轉(zhuǎn)。離子交換膜將電極室與電泳室隔開。電泳結(jié)束，可用真空多頭取樣裝置一次快速將每個隔室內(nèi)聚焦好的樣品取出，防止區(qū)帶混合。Bio－rad商品，可上樣30-50ml，總上樣蛋白質(zhì)可達(dá)3克。2-DElectrophoresisIn2-Delectrophoresis,proteinsareseparatedinthefirstdimension,accordingtotheirisoelectricpoints等電點(diǎn),byIEF,andintheseconddimension,accordingtotheirmolecularweights分子量,bySDS.Theresult,afterstaining,isanumberofspotsdistributedintwodimensionsoverthegel.pH3------10(1)IEF等電聚焦電泳(2)SDS(3)染色脫色JuangRH(2004)Proteomics■雙向電泳操作流程：2-DElectrophoresisCapillaryelectrophoresis(CE)TheelectrophoreticmigrationvelocityThevelocityoftheelectroosmoticflow(電滲流)當(dāng)pH>3時，毛細(xì)管內(nèi)壁表面帶負(fù)電荷，因靜電引力會吸引周圍液體帶有相反電荷（正電荷），形成雙電層。當(dāng)液體兩端施加電壓時，就會發(fā)生液體相對于固體表面的移動（一般移向陰極），這種現(xiàn)象叫電滲現(xiàn)象。ElutionOrderNeutralsarenotseparated!FreeFlowElectrophoresisFreeFlowElectrophoresis(FFE)isanelectrophoresisprocedureworkingcontinuouslyintheabsenceofastationaryphase(orsolidsupportmaterialsuchasagel).Itseparatespreparativelychargedparticlesranginginsizefrommoleculartocellulardimensionsaccordingtoth