GE公司培訓(xùn)講義—凝膠過濾層析技術(shù)

1、 #/GE/Iimaginationatwork /GE/ #/GE/Iimaginationatwork #/GE/ #/GE/Iimaginationatwork #/GE/Whatisgelfiltration凝膠過濾定義？Gelfiltration(GF)isasimpleandreliablechromatographicmethodforseparatingmoleculesaccordingtosizeandshape凝膠過濾層析是根據(jù)分子大小和形狀的差異進(jìn)行分離的一種簡單可靠的層析技術(shù)。 #/GE/Iimaginationatwork #/GE/ #/GE/Iimaginati

2、onatwork #/GE/Gelstructure填料結(jié)構(gòu)凝膠過濾分離原理：空間排阻 #/GE/Iimaginationatwork #/GE/AGAROSE瓊脂糖凝膠2.Sampleapplied.3.Buffer(mobilephase)andsample4.Largemoleculesleavethecolumnfirstfollowedbysmallermoleculesinorderofsize.Moleculeslargerthanthematrixporespassstraightthrough.movethroughcolumn.Moleculesdiffuseinandou

3、tofmatrixAgoodgelforgelfiltrationcontainsabout95%water凝膠過濾填料的95%為水-poresfilledwitheluent孔內(nèi)充滿洗脫液-carefullycontrolledsizerange孔徑精密控制-chemically/physicallyinert(lackofadsorptiveproperties)惰性iTi2cinationatwork /GE/Iimaginationatwork /GE/Stericexclusionleadstoearlyelution體積排阻程度決定洗脫位置Moleculeseluteinorder

4、ofsize.不同分子按照大小順序洗出Thelargestmoleculeselutefirst;thesmallestoneselutelast.大分子先洗出，小分子后洗出ThevoidvolumeV夕卜水體積o非常大的夕子在外水體積洗出,V。naen1fo111%1fIvolumeDistributioncoefficient,K分配系數(shù)dKv-v=R0dV-V-Vc0sV-VR0ViVr=Vo+Kd*Vi=保留體積/洗脫體積=排阻體積/外水體積=層析柱幾何體積=固相凝膠所占的體積=孔內(nèi)體積/內(nèi)水體積=V-V-V-V:外水體積+能進(jìn)入的那部分內(nèi)水體積-K：:溶質(zhì)分子能夠進(jìn)入的內(nèi)水體積（內(nèi)水

5、體積）占總的內(nèi)水d體積的比例K較難測定，常用Kav代替Separationvolumes分離體積4-EIIVVVVR1R2R3R4Fdiial：slolh=p:rcvoidvolumeVvolumeofthe.gelmatrixVporevolumeVvoidvolume外水體積retentionvolumeorelutionvolume保留體積/洗脫體積totalliquidvolumeofthebed總液體體積innerporevolume=V-V-V內(nèi)孑L體積/內(nèi)水體積totalgeometricvolumeofthecolumn柱體積SVtandV總液體體積和柱體積tcThecoeff

6、icientKavKv-vROav一v-Vc0-與柱子大小和填裝無關(guān)-可以用于比較不同的純化結(jié)果Kav容易得到，也更有實際意義 /GE/Iimaginationatwork #/GE/K應(yīng)該在0和1之間avKforverylargeandverysmallmolecules #/GE/Iimaginationatwork #/GE/ #/GE/Iimaginationatwork #/GE/非常大的分子在V處洗脫V=V0R0KV-V=R0=哄=0avV-VV-Vc0c0elutionvolumewhenK=1elutionwi岀K1Adsorptionhasoccurred.非常小的分子在V處

7、洗脫V=VRtKV-VV-V1avv-vv-vc00i扌vohlumeelutionwithK0Seriousprobltm!曆+流動相中需要加入150mMNaCl！ #/GE/Iimaginationatwork #/GE/ #/GE/Iimaginationatwork #/GE/Calibrationcurves校準(zhǔn)曲線Selectivitycurves選擇性曲線 #/GE/Iimaginationatwork #/GE/ #/GE/Iimaginationatwork #/GE/VversusthelogarithmofmolecularmassKversusthelogarithmo

8、fmolecularmassav：wSelectivitycurves選擇性曲線Exclusionlimit排阻極限 #/GE/Iimaginationatwork #/GE/不能進(jìn)入填料孔的最小的分子量 #/GE/Iimaginationatwork /GE/ExclusionlimitAllmoleculesbiggerthanthiseluteinthevoidvolume.分子量大于此的分子均在外水體積洗出rlog(M)r #/GE/Iimaginationatwork #/GE/ #/GE/Iimaginationatwork #/GE/iminationatwork /GE/Iim

9、aginationatwork /GE/Fractionationrange分級范圍K=0.1toK=0.7的范圍內(nèi)，選擇性曲線為直線avavK1av0.70.10fractionationrange填料分級范圍log(M)rResolutionfactor,R分辨率sV-V21(W+W)/212兩峰相對于其峰寬而言，分開的有多遠(yuǎn)；：iff*選擇最佳的凝膠(highselectivity)SuperdexPeptideSuperdex75Superdex200SephacrylS-100HRSephacrylS-200HRSephacrylS-300HRSephacrylS-400HRCont

10、ent內(nèi)容Principlesofgelfiltration凝膠過濾原理Optimize:Howtogettheexpectedresults優(yōu)化Productsandapplications產(chǎn)品和應(yīng)用Practicalconsiderations實際需考慮的因素Summary總結(jié)Resolution:dependson分辨率取決于selectivity選擇性andefficiency柱效highselectivitySelectivity選擇性:峰分離的量度高選擇性可以達(dá)到基線分離高選擇性可以彌補(bǔ)低的柱效highefficiencylowefficiency-峰體積有可能增加Efficien

11、cy柱效:峰寬的量度柱效越高峰越窄丿lowselectivityhighefficiencyi-lowefficiency高柱效需要良好的柱填裝技術(shù)高柱效有時可彌補(bǔ)選擇性不足FractionationRange分級范圍 /GE/iimccinationatwork #/GE/iimccinationatworkShapeeffects形狀的影響Resultsdependonselectivity分離結(jié)果與選擇性有關(guān) #/GE/iimccinationatwork #/GE/iimccinationatwork #/GE/iimccinationatwork #/GE/iimccinationa

12、tworkAuSephacrylS-3001Ok-15OOk適合較大分子CytidineSephacrylS-200I加增限極阻排bsaCytCb-LigG5k-250k最佳1k-100k適合較小分子變性蛋白Agelhasdifferentfractionationrangesfornative,globularproteinsanddenaturedproteinsinrandomcoil.對于形狀不同的分子，某填料的分級范圍不同 #/GE/iimccinationatwork #/GE/iimccinationatwork #/GE/iimccinationatwork #/GE/iimc

13、cinationatworkShapeeffects形狀的影響K1avglobularproteinsMoleculeswithdifferentshapeshavedifferentselectivitycurves.對于形狀不同的分子，有不同的選擇性曲線。Efficiency柱效Efficiencydependson取決于：flowrate流速particlesizeofmatrix粒徑大小particlesizedistributionofmatrix粒徑分布packingqualityofthecolumn層析柱的裝填samplevolumeandviscosity樣品體積和黏度 #/

14、GE/iimccinationatwork #/GE/iimccinationatwork #/GE/iimccinationatwork #/GE/iimccinationatwork #/GE/iimccinationatwork #/GE/iimccinationatwork #/GE/iimccinationatwork #/GE/iimccinationatworkPeakwidthdependsonparticlesize粒徑影響柱效Resolutiondependsonsamplevolume進(jìn)樣體積影響柱效 #/GE/iimccinationatwork #/GE/iimcci

15、nationatwork #/GE/iimccinationatwork /GE/iimccinationatworkSuperdexTPeptide13-15pmSuperdex30prepgrade24-44pmSuperdexPeptideHighPerformancecolumn10/30_24ml /GE/IiTcginationatwork #/GE/IiTcginationatwork2xSuperdexPeptideHighPerformanee1xSuperdexPeptideHighPerformaneecolumn10 x300mmi.dxbedheight0Resolu

16、tiondependsonsampleviscosity黏度影響柱效樣品黏度過高，分離度下降.HaemoglobinandNaCl,viscositychangedbyaddingdextranResolutiondependsoncolumnlength柱高影響分辨率Increasingcolumnlengthincreasesresolution柱高加長，分辨率提高 #/GE/IiTcginationatwork #/GE/IiTcginationatwork #/GE/IiTcginationatwork #/GE/IiTcginationatworkContent內(nèi)容Principle

17、sofgelfiltration凝膠過濾原理Optimize:Howtogettheexpectedresults優(yōu)化Productsandapplications產(chǎn)品和應(yīng)用Practicalconsiderations實際需考慮的因素Summary總結(jié)Mainpurificationtasks主要應(yīng)用Groupseparations組分離-desalting,bufferexchange,removingreagents脫鹽/換緩沖液Fractionationofproteinsandpeptides精細(xì)分離-complexsamples,monomer/dimer聚集體去除Estimati

18、onsize測定分子量Characterisation:sizehomogeneity蛋白鑒定：均一性 #/GE/IiTcginationatwork #/GE/IiTcginationatwork #/GE/IiTcginationatwork #/GE/IiTcginationatworkGroupseparations組分離Fractionation精細(xì)分離 #/GE/IiTcginationatwork #/GE/IiTcginationatworkDesaltingproteins蛋白質(zhì)快速脫鹽Separatingdimerandoligomersfrommonomer去除蛋白聚集體

19、 #/GE/IiTcginationatwork #/GE/IiTcginationatwork #/GE/IiTcginationatwork #/GE/IiTcginationatworkColumn:Sample:Load:Buffer:c0.05.010.015.020.025.0mlColumn:Sample:Samplevolume:Elutionbuffer:Flowrate:System:Detection:Hitrapdesalting5mlHSA,2mg/ml,0.8,1.3,1.7,2.2ml(16-26%Vt)NaCl0.5M組分離:柱高60cm,進(jìn)樣體積較大(和！(2

20、rfrtTtnhueriSuperdexPeptide-分離范圍100-7000Da-高化學(xué)穩(wěn)定性,pH1-14,兼容有機(jī)溶劑（乙腈+TFA）-和RPC銜接，用于peptide的高效分離純化AggregatesRemoval聚集體去除iIKrv敦I/*、1SDS*加iGd=覺1TIJRequirement:Monomer95%r.*=4-1j41-bl!4gni心nd.*;g?HMiti：hM加ffmraMdrer：丫:恢卜iJHfcocyui|hi.:;iSuperdex20010/300GL；址;4：I1K4.1-igRapidpuritycheckofr-protein重組蛋白純度快速檢

21、測Recombinantprotein17kDa.Column:Superdex755/150GLBuffer:PBSpH7.4Flowrate:0.3ml/minSamplevolume:4plAnalysistime:12min/run樣品純度檢測非?？炜梢源致怨罍y純度精確的定量分析請選擇10/300GLSephacrylHR-由葡聚糖交聯(lián)N,N亞甲基二乙酰胺形成-理化性質(zhì)穩(wěn)定-系列全，成本低類病毒顆粒的純化(VLP)HiPrep16/60SephacrylS-500HR沖啊卿in科豳腳Collaborationw諾Novavax #/GE/Iimaginationatwork /GE/S

22、uperoseSepharose-高度交聯(lián)的瓊脂糖-分離范圍廣-理化性質(zhì)穩(wěn)定id&4滯訕犢口rbr-交聯(lián)的瓊脂糖-理化性質(zhì)穩(wěn)定-FastFlow填料流速快：250-300cm/hSepharose4FF:60kD20MDSepharose6FF:10kD4MD-常用于疫苗領(lǐng)域 #/GE/Iimaginationatwork #/GE/ #/GE/Iimaginationatwork #/GE/750kultrafiltrationultra-/dafiltration1AEX:QSepharoseFFrose4FF陰離子父換CaptoadhereSepharose4FF終產(chǎn)品蛋白殘留1%Cel

23、lculturesupernatantInflu.&RabiesVaccineProduction流感&狂犬等病毒類疫苗的生產(chǎn)purifiedhumaninfluenzavirus關(guān)3多糖疫苗：兩步層析代替酚抽提 #/GE/Iimaginationatwork #/GE/ #/GE/Iimaginationatwork #/GE/多糖疫苗的檢定超螺旋質(zhì)粒pDNA純化 /GE/Iimaginationatwork #/GE/藥典CP2005：用瓊脂糖-4B或CL-4B凝膠過濾法測定，KD值應(yīng)0.40（即retention2處的Kd2值0.40）。值表示相對分子量，多糖抗原分子量要大于10kd才具

24、有免疫原性。Clarifiedalkalinelysate裂解液.HiTrap26/10Sepharose26mmx100mmVt:53mlRNA-removalHiTrapPlasmidSelectXtra16mmx25mmVt:5mlSupercoiledpDNAcaptureHiTrapSOURCE30Q16mmx25mmVt:5mlSupercoiledpDNApolishingPurifiedsupercoiledpDNA純化的pDNAPlasmidSelectXtraStarterKit6g一-1一廠J*! #/GE/Iimaginationatwork #/GE/ #/GE/Ii

25、maginationatwork #/GE/imaginationatwork #/GE/iminationatwork /GE/iminationatworkSephadex-由葡聚糖交聯(lián)異表氯醇形成的多孔結(jié)構(gòu)-SephadexG10:100-700Da多肽純化,oligo脫鹽，抗生素聚集體質(zhì)控分析-SephadexG25:1k-5kDaMW5kD蛋白快速脫鹽隅Bufferexchangeusing使用脫鹽柱快速脫鹽HiPrep26/10DesaltingColumnSample:BSAdissolvedin50mMpiperazine,0.5Msodiumchloride,pH6.2Col

26、umn:HiPrep26/10Desalting_53mlBuffer:20mMsodiumphosphate,0.15Msodiumchloride,pH7.0System:AKTAprime,20ml/min #/GE/iminationatwork #/GE/iminationatwork #/GE/iminationatwork #/GE/iminationatworkContent內(nèi)容Principlesofgelfiltration凝膠過濾原理Optimize:Howtogettheexpectedresults優(yōu)化Productsandapplications產(chǎn)品和應(yīng)用Pract

27、icalconsiderations實際需考慮的因素Summary總結(jié)4keyquestions4個問題1.Whatistheaimoftheexperiment?實驗?zāi)康模?Highresolutionfractionation精細(xì)分離or還是-Groupseparation(desalting,bufferexchange)?組分離，緩沖液置換？Highresolutionfractionatio精細(xì)分離:Superdex,Superose,SephacrylGroupseparation組分離:Sephadex #/GE/iminationatwork #/GE/iminationatw

28、ork /GE/iminationatwork #/GE/iminationatwork4keyquestions4個問題2.Whatisthemolecularweightofthetargetprotein/contaminants?目標(biāo)蛋白和雜質(zhì)的分子量Chooseagelfiltrationmediumwiththefractionationrangethatcoversthemolecularweightvaluesofinterestinyoursampleandthatgiveshighestdifferenceinsizeoftargetprotein/contaminants

29、/salt選擇分級范圍能包含目標(biāo)蛋白，且使雜質(zhì)和目標(biāo)蛋白的分離度最大的填料4keyquestions4個問題3.Howmuchsamplearetheyloading?進(jìn)樣量是多少?？Desalting脫鹽25%CVPD-10(gravity)loadingcapacity2.5mlHiTrap5mlloadingcapacity1.5mlHiPrep53mlloadingcapacity15mlFractionation-polishing精細(xì)純化l-analyticalcolumn分析1%CV-10/300loadingcapacity250l-5/150loadingcapacity50lml-prepa