凝胶层析.docx
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凝胶层析.docx
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凝胶层析凝胶层析GelFiltrationChromatographyBio96zoumi20090300691.Objectives1)Tounderstandtheprincipleofgelchromatographyanditsapplication.2)Bymeasuringtheproteinmolecularweighttraining,initiallymastergelchromatography.2.principlesGelchromatography,alsoknownasexclusionchromatography,gelfiltration,molecularsievechromatography,filtrationorinfiltration.Itiswidelyusedinseparation,Purification,concentrationanddesaltingofbiologicalmacromolecules,andsoon,whilethedeterminationofproteinmolecularweightisalsooneofitsimportantapplications.Gelisakindofthree-dimensionalnetworkstructurewasporousandthebead-shapedinsolubleparticulatematerial.Whenusingittoseparatesubstances,itismainlyaccordingtothedifferentradiiofproteinmolecules(near-spherical)withdifferentexclusioneffects.Thatis,itisbasedonthemolecularsizeofthephysicalpropertiesoftheseparationandpurification.Forcertaintypesofgel,somemoleculescannotentertheparticles,andcanonlyflowalongtheoutercolumn;whileanumberofsmallmoleculesarenotexcluded.Theyarefreetospread,infiltratingintothegelinsidethesieve,andlateristakenout.Thesmallermolecules,thedeeperinsideintothegel,themoredistancetheygo,sothesmallmoleculesarethelasttoflowout.Somemedium-sizedmoleculesbetweenmacromoleculesandsmallmoleculescanonlyenterthepartofthelargergelpores,whichispartlyexcluded,sothesemoleculescomeoutbetweenlargeandsmallmolecules.Thissampleaftergelchromatography,themoleculeswillbeinaccordancewithbothlargeandsmallintheflowingordertoachievethepurposeofseparation.Foranykindofcompoundsseparatedbygel,theexclusionchromatographycolumnsareintherangeof0100%,anditsrankeddegreeofbeingexcludedcanbedescribedbyKav(isolatedcompounds,includingouterwaterandexternalwatervolumeintheproportionalrelationship).AsforKavandthetotalvolumeofgelcolumnbed(Vt),outerwatervolume(V0),andtheelutionvolume(Ve)thereisaformulalikethis:
Kav=(Ve-V0)/(Vt-V0).Chromatographyunderthefixedconditions,VtandV0isaconstantvalue,Vechangeswiththechangesofthemassofthemoleculestobeseparated.Thelargerthemolecularweightis,thesmallerthesmallerVevalueandKavvaluewillbe.Onthecontrary,thesmallerthemolecularis,thebiggertheVevalueandKavvaluewillbe.Kavisimportanttoestimatetheseparationeffectandthemassofproteinmolecules.Inthesamechromatographicconditions,themoredifferenttheKavvaluesofseparatedmaterialis,thebetterseparationeffectwillbe.Onthecontrary,separationefficiency,otherwisethemoleculescantbeseparatedortheeffectisverybad.Intheactualexperiment,wemeasuredaVt,V0andVevalues,andthencalculatetheKav.Foraparticulartypeofgel,inthecontextofacertainmolecularweight,Kav,andlgMr(Mrindicatedthatsubstancemolecularweight)intoalinearrelationship:
Kav=-blgMr+CThesamecanbe:
Ve=-blgMr+CWhereb,Careconstant.Namely,VeandlgMrhasalinearrelationship.WecanisolateKnown-weightmolecularproteinsinavarietyofgelcolumn,andinaccordancewiththeabove-mentionedlinearrelationshipbetweentheplottedstandardcurves,andthenmeasuretheotherunknownproteinmolecularweightusingthesamegelcolumn.3.Equipmentandreagents1)Equipment
(1)Glasschromatography
(2)DHL-BPCconstantpump(ShanghaiJingBranchedIndustrialCo.,Ltd.)(3)BSZ-100automaticpartofthecollector(ShanghaiQingpuInstrumentFactoryHuxi)(4)8823BUVdetector(5)UNICCOUV-2102Cuv-visspectrophotometer(6)TYPE3057PortableRecorder(SichuanNo.4InstrumentFactory)(7)TB-600gradientmixer(ShanghaiUnityBiochemicalInstrumentFactory)(8)100mlreagentbottle(9)1000mlgraduatedcylinder(10)250mlbeaker(11)50ml,100mlbeaker(12)10mltest-tubescale2)ReagentsStandardproteinBovineserumalbumin:
Mr=67000(ShanghaiBiochemistry).Eggalbumenprotein:
Mr=45000(U.S.SIGMAcompany).Lysozyme:
Mr=14300.UnknownproteinsamplesElute0.025mol/LKCL0.1mol/LHACBluedextran20004.Experimentprocedures1)GelswellingWeigh7gSephadexG-75inthe250mlbeaker,add100mlelate,swellatroomtemperaturefor23d,repeatedlyPourremovethefineparticles,andthenplacegelinvacuumtocasttheairbetweentheparticles,thenintoboilingwaterbathtoboil23h(torunawayInternalairoftheparticles,andtosterilized).2)Loadingthecolumn
(1)Takeacleanglasschromatographycolumnandverticallyfixeittothemetalframeworkstage.
(2)Determiningthetotalvolumeofgelcolumn(Vt)Makeamark5cmawayuptothecolumnend,closethecolumnoutlet,adddeionizedwater,opentheoutlet.Whentheliquidleveldroptothemark,closetheoutlet.Thenusethemeasuringcylindertoacceptthede-ionizedwater(surfacedowntotheglasssieve),readthecolumn.OutofthetotalvolumeofthecolumnbedvolumeshallbeVt.WecanalsomeasuringVtafterhavingdonetheunknown-protein.(3)addingelatetothecolumn(about1/3columnbedheight),slowlypourtthickslurrytothecolumnuntilthegelbecomes1cm2cmhighandthenopentheoutlet,theflowratetendstobeabout36ml/10min.whenthegelgoestothemark,andtheloadingiscompleted,becarefulthattheloadinggelcannotbelayered.Andthenclosetheoutlet,putitasideforamoment,untilthegeldeposits,thenconnectittotheconstant-current.Use12timesofthebedvolumeofelatetobalancecolumn,sothatmakesureofthestability.3)DeterminingV0andtheVeoftheunknownproteinDrawawaytheelateonthesurface(mustnotupsetthesurface,filterpaperornylonnetcanbecovered).Opentheoutlet,sothatresidualliquidisreducedtogluefacialcut(butnotletitdry).Closetheoutlet.Suckupwithafinedropper0.5mlmixtureof(4.5mg/ml)blue-color-glucan-2000andunknownprotein(5mg/ml)mixture,carefullyadditcirclingaroundthecolumnwall(2mmabovethegelsurface),andthenopentheoutlet(Startcollecting!
),Afterthesolutionintothegelbed,Closetheoutletwithalittleeluentaddingtothebed,fillthetopwitheluent.Startelutingatthespeedof56ml/10min.automaticcollectorforeachtubeissetat5mincollection.FinallymakeelutioncurveFinallymadeelutioncurve.CollectionandplanningbasedonexpenditurefromtheCanadianKindofbeginningtotheelutionconcentrationofbluedextranelutionvolumeshallbethehighestpointoftheV0.Note:
BlueDextranwashdownAfterwards,usetheelate(12timesthebedvolume)tobalance,preparingforthenextstep4)Thestandardcurve
(1)Thestandardproteinsolutionwiththeeluentispreparedforclassuse,thesolutionofvariousconcentrationsofthethreeproteinsare:
bovineserumAlbumin(5mg/ml),chickeneggalbumin(8mg/ml),lysozyme(4.5mg/ml).
(2)Accordingtotheoperationmethod(3)addthestandardprotein0.5mlofthesolution56ml/10minandcollecteluent.(3)ByusingultravioletspectrophotometerdetermineA280tubebytubeanddecidethepeakpointofproteinelution,andthenmeasuredproteinelutionvolumeVe.Notethataftertheexperimentiscompleted,getallthegelIrecycledforthenexttesttouse.Itsstrictlyprohibitedtodiscard,orpourthegelintothePool.5.Records1)rawdataEluteSpeed:
0.6ml/minAutomatedportionofthecollectorspeed:
(forthefirsttime)5min/tube,(second)3min/tubeRecordingPapersspeed:
2cm/hUVDetector:
(forthefirsttime)0.5A,(second)0.2AThetotalvolumeofgelcolumns:
Vt=86.0mlThebluedextranelutionvolume(externalwatervolume):
V0=31.3mlUnknownproteinelutionvolume:
Vg=51.4mlStandardproteins:
Ve1=32.2mlVe2=41.2mlVe3=65.6ml2)AbsorbanceA280DatanumberA280VNumberA280V10160.09320.002170.08030.002180.12440.008190.27450.003200.45460.003210.53151.4ml7-0.003220.4368-0.003230.26690.001240.125100.011250.026110.001260.000120.39727-0.017131.17031.3ml140.590150.188Table2-1280nmUVabsorbanceofsample1numberA280VnumberA280V10.000260.05920.001270.0653-0.00128-0.0224-0.00329-0.0485-0.00230-0.04960.003310.04770.001320.02580.002330.1029-0.001340.18410-0.001350.25111-0.001360.272120.001370.27665.6ml130.002380.2140.011390.131150.004400.059160.011410.101170.142420.07180.20632.2ml430.123190.173440.031200.141450.019210.124460.004220.082470.083230.16541.2ml480.019240.129490.005250.116500.003Table2-2280nmUVabsorbanceofsample2标准蛋白洗脱曲线Figure2-1Standardproteinelutioncurve6.DataProcessing1)ThetotalvolumeofgelcolumnVt=86.0ml2)ExternalwatervolumeV0andtheunknownproteinelutionvolumeVeFigure2-2AbsorbanceGraphofsample1ThefirstelutionpeakisfortheexternalwatervolumeV0.ThesecondpeakisforunknownproteinVe.Betweenthecolumnandtheautomaticalcollector,thereisabout5mlliquid.Whencounttheelutionvolume,itshouldbesubtracted.V0=36.0-5.0=31.0(ml);Vunknown=51.4-5.0=46.4ml.3)StandardproteinelutionvolumeVeFigure2-3AbsorbanceGraphofsample2Thefirstthreepeaksareasfollowing:
bovineserumalbuminelutionpeak,eggalbuminelutionpeak,lysozymeelutionpeak.Bovineserumalbumin:
Mr=67000,albumin,:
Mr=45000,Lysozyme:
Mr=14300.Measured:
Ve(bovineserumalbumin)=32.2-5.0=27.2ml;Ve(eggalbumin)=41.2-5.0=36.5ml;Ve(lysozyme)=65.6-5.0=60.6ml4)TheproteinintheeffectivedistributioncoefficientKavandtheunknownproteinmolecularweightcalculationproteinMrLgMrVt/mlV0/mlVe/mlKavbovineserumalbumin670004.82686.026.327.20.015eggalbumin450004.65336.50.171lysozyme143004.15560.60.575unknownprotein46.40.337Figure2-4Kav-lgMrgraghoftheknownproteinsPuttheunknownproteinKav=0.337intothefittingformula.SowecanfetlgMr=4.311,Mr=27803.53.Figure2-5Ve-lgMrgraghGettheunknow
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