亲和原理篇.pptx

内容1.原理什么是亲和层析?亲和层析是通过生物分子的生物相互作用进行生物分子分离的一项液相分离技术。1.平衡平衡2.上样3.结合/洗涤4.洗脱平衡层析柱至可以结合样品的状态凝胶基架特异结合配基亲和层析亲和层析结合目标洗涤杂质在可结合状态结合样品1.平衡2.上样上样3.结合结合/洗涤洗涤4.洗脱亲和层析的主要步骤平衡：让亲和填料处于可以结合目标物的状态上样：将样品通过填料，洗出不结合的杂质洗脱：解吸附，洗脱下目标产物恢复：重新平衡使填料处于可以结合目标物的状态亲和层析EquilibrationSample applicationWashElutionRe-equilibration亲和层析的主要步骤亲和层析亲和层析特点亲和层析特点容易纯化一些别的方法不能分离的蛋白比如：从变性的蛋白中分离天然蛋白及其不同功能的亚型经常一步可用大于95%的纯度从大量杂质中分离高纯度的目标物质可以特异性的去除杂质非常快速亲和层析2.凝胶及其应用配基:特异性的和目标物结合的基团基架:可以和配基耦合的内部填料耦合:配基和基架之间的共价结合结合:配基和目标产物之间的结合亲和层析中的术语亲和填料金属螯合：金属螯合：可反复螯合不同金属，再用已结合依赖不同金属的蛋 白，一种介质可做多种纯化工作活化偶联：最常用的是NHS activated 4 Fast Flow 和CNBr activated 4 FF小配体亲和：纯化抗凝血酶III，凝血因子脂酶等抗体亲和：抗体亲和：颜料亲和：纯化白蛋白，干扰素等外源凝集素亲和：刀豆球蛋白A-多糖，糖蛋白等核酸亲和金属螯合可用反复螯合不同金属，再结合依赖不同金属的蛋白。（His组氨酸、Cys半光氨酸、Try色氨酸）填料类型：IMAC Sepharose：固定化金属离子亲和层析填料，基架已经活化好了，可用于直接耦合金属离子。Ni Sepharose：已经耦合了Ni离子，可以直接用于纯化His蛋白,载量极高，脱落极低。Chelating Sepharose：亚氨双乙酸耦合的填料，可用结合不同的金属离子，脱落低。（详细介绍请参考Tag-protein.ppt）NHS-activated Sepharose High Performance12-atom hydrophilic spacer arm to couple via amino groups.NHS-activated Sepharose 4 Fast FlowAs above.CNBr-activated Sepharose 4 Fast FlowCoupling via primary amino groups.EAH Sepharose 4B10-atom spacer arms to couple via amino groups.ECH Sepharose 4B9-atom spacer arms to couple via carboxyl groups.Epoxy-activated Sepharose 6B12-atom hydrophilic spacer arm to couple through hydroxyl,amino or thiol groups.Activated Thiol Sepharose 4B10-atom spacer arm for reversible coupling through freethiol groups.Thiopropyl Sepharose 6B4-atom hydrophilic spacer arm for reversible coupling of proteinsand small thiolated ligands through thiol groups.Also reacts withheavy metal ions,alkyl and aryl halides and undergoes additionreactions with compounds containing C=O,C=C and N=N bonds.最常用的是NHS activated 4 Fast Flow 和CNBr activated 4 FF填料类型：活化偶联亲和填料耦合方法1(2)Chemical groupLength ofStructure ofProducton ligandspacer armspacer armProteins,peptides,amino acidsamino10-atomHiTrap NHS-activated HPNHS-activated Sepharose 4 Fast FlowNoneCNBr-activated Sepharose 4BCNBr-activated Sepharose 4 Fast Flow10-atomECH Sepharose 4Bcarboxyl11-atomEAH Sepharose 4Bthiol4-atomThiopropyl Sepharose 6B10-atomActivated Thiol Sepharose 4B 12-atomEpoxy-activated Sepharose 6BONOHOONOOONOHOHOONOHNH2OSOHSNNNSOSNNOHOOOHOOOOOHO活化偶联亲和填料Chemical groupLength ofStructure ofProducton ligandspacer arm spacer armSugarshydroxyl12-atomEpoxy-activated Sepharose 6Bamino10-atomHiTrap NHS-activated HP10-atomECH Sepharose 4B12-atomEpoxy-activated Sepharose 6Bcarboxyl11-atomEAH Sepharose 4BOOOOHOONOHOONOOONOHOHOPolynucleotidesaminoNoneCNBr-activated Sepharose 4BCNBr-activated Sepharose 4 Fast Flowmercurated base4-atomThiopropyl Sepharose 6BCoenzymes,cofactors,antibiotics,steroidsamino,carboxyl,thiol or hydroxyl use matrix with spacer arm(see above)OOOOHOONOHNH2OSOHSN耦合方法2(2)活化偶联亲和填料ProductSpacerCouplingMaximumCommentsarmconditions operating flowHiTrap NHS-activated HP 10-atompH 6.59,1530 min.,4 ml/min Pre-activated medium+4 C room temp.(1 ml column)for coupling via 20 ml/min primary amine group(5 ml column)of a ligand.Prepacked 1 ml and 5 ml columns.NHS-activated 10-atompH 69,216 hours,300 cm/h*Supplied as a Sepharose 4 Fast Flow+4 C room temp.suspension ready for column packing.活化偶联亲和填料ProductLigand CompositionpH stability*Mean particle density sizeHiTrap 10 moles/ml6-aminohexanoic acidShort term 31234 mNHS-activated HP linked by epoxy coupling to Long term 312highly cross linked agarose,terminal carboxyl group esterified with NHS.NHS-activated 1623 moles/ml As aboveShort term 31390 mSepharose 4Long term 313Fast Flow NHS-activatedOOOH+RCO52222CH(CH)NHNHOOCHCHNOOOHCR5222CH(CH)NHNH+HOOCHCHNOSepharoseSepharose活化偶联亲和填料NHS-activatedReady in less than 15 minutesProtein coupled(mg)Protein added(mg/1 ml column)4030201020406080100Mouse IgG on HiTrap NHS-activated HP,1 ml20406080100 mlLane 1.Eluted material,non-reducedLane 2.LM W Calibration Kit,reduced1 2Mr97 00066 00045 00030 00020 10014 0001 2Mr97 00066 00045 00030 00020 10014 0002.01.0A280 nmFlow through materialNHS-activated活化偶联亲和填料ProductSpacerCouplingMaximumCommentsarmconditionsoperating flowCNBr-activatedNonepH 79,216 hours,400 cm/h*Supplied as a freeze-Sepharose 4 Fast Flow+4 C room temp.dried powder.CNBr-activatedNonepH 810,216 hours,75 cm/h*Supplied as a freeze-Sepharose 4B+4 C room temp.dried powder.ProductCompositionBinding capacitypH stability*Mean particleper ml mediumsizeCNBr-activatedCyanogen bromide reactsa-chymotrypsinogen,Short term 31190 mSepharose 4 Fast Flowwith hydroxyl groups on1326 mgLong term 311Sepharose to give aCNBr-activatedreactive product fora-chymotrypsinogen,Short term 211Sepharose 4Bcoupling ligands via2560 mgLong term 211primary amino groups orsimilar nucleophilic groups.活化偶联亲和填料CNBr-activatedCNHisoureaNHRCNBrRNH2OOHOHHOSepharoseSepharoseCNBr-activated活化偶联亲和填料CNBr-activated Sepharose 4B washed at pH 3 and transferred to 0.1 M NaHCO3,pH 8.3 for pre-hydrolysis.Samples removed after different times and tested for coupling activity towards a a-chymotrypsinogen(A)and glycyl-leucine(B).glyleuTime of pre-hydrolysis,hours02134204060010024ABAB80Coupled substance,%a-chymotrypsinogenA280 nm0.5024 hours40 minTimenative gp120Lectin coupled to CNBr-activated Sepharose 4 Fast Flow活化偶联亲和填料CNBr-activatedProductSpacerSubstitutionCouplingMaximum Commentsarmper ml matrixconditionsoperatingflowEAH Sepharose 4B10-atom711 molespH 4.5,1.524 hours,75 cm/hCouple ligands amino+4 C room temp.containing free groupscarboxyl groups.Supplied as a suspension ready for use.ECH Sepharose 4B9-atom1216 molespH 4.5,1.524 hours,75 cm/hCouple ligands carboxyl+4 C room temp.containing free groupsamino groups.Supplied as a suspension ready for use.ProductCompositionpH stability Mean particle size EAH Sepharose 4BCovalent linkage of 1,6-diamino-hexaneShort term 31490 mby epoxy coupling creates a stable,Long term 314uncharged ether link between a 10-atomspacer arm and Sepharose 4B.ECH Sepharose 4BCovalent linkage of 6-aminohexanoic acidShort term 31490 mby epoxy coupling creates a stable,Long term 314uncharged ether link between the 9-atom spacer arm and Sepharose 4B.活化偶联亲和填料ECH&EAH Sepharose SepharoseOHCOOH5222CH(CH)NHO CHCHOHNH62222CH(CH)NHO CHCHSepharosePartial structuresECH Sepharose 4BEAH Sepharose 4BPartial structures活化偶联亲和填料ECH&EAH Sepharose ProductSpacerSubstitution CouplingMaximum Commentsarmper ml matrixconditionsoperatingflowEpoxy-activated12-atom1940 molespH 913,16 hours 75 cm/hSupplied as aSepharose 6Bepoxy groupsseveral days,freeze-dried+20 +40 C powder.CompositionpH stability Mean particlesizeSepharose 6B reacts withShort term 21490 m1,4 bis-(2,3 epoxypropoxy-)Long term 214butane to form a stableether linkage.OH422222CHCH(CH)OOOO CHCHCHCHSepharosePartial structureEpoxy-activated Sepharose 6B活化偶联亲和填料Carbonate/bicarbonate buffers pH 911NaOH solution pH 1214.Ligand concentrations:5 and 20 mg/mlN-acetyl-D-galactosamine to Epoxy-activated Sepharose 6B20 mg/ml5 mg/mlpH910111213255075010014Coupled ligand moles/g conjugateGlycyl-leucine to Epoxy-activated Sepharose 6BpH 10.5,+40 CpH 11.0,+45 CTime hours0816243225507501004048Coupled glycylleucine,%活化偶联亲和填料Epoxy-activated Sepharose 6BMixed disulphide formation(1),reversible by reducing agents such as dithiothreitol(DTT)(2).Mixed disulphide formation with 2,2-dipyridyl disulphide:2-thiopyridyl derivative for covalent chromatography(3).Reaction with heavy metals and derivatives e.g.p chloromercuribenzoate(4):mercaptide formation.Treatment with alkyl or aryl halides:thioether derivatives(5).Addition reactions(6)with C=O,C=C and N=N bonds.Thiopropyl Sepharose 6BCHCHCONHCovalentDTTPDSOOSSHNSSCOOHHgS2CHOCHSNO2S22SROHCHSNHRRNSRSS112234556666Chromatography活化偶联亲和填料小配体亲和填料填料上预先已经耦合了不同类型的小配体，用以结合不同的特异性激酶、重组蛋白或者其他活性物质等。填料类型：Glutathione Sepharose:填料上已经耦合了谷胱甘肽，可用结合含S-转移酶的重组蛋白。Heparin Sehparose：纯化抗凝血酶III，凝血因子脂酶等。Benzamidine Sepharose：专一性的结合丝氨酸蛋白酶、胰蛋白酶、类胰蛋白酶等。其他填料：纯化不同的蛋白。（详细Glutathione Sepharose信息请参考Tag-protein.ppt）Binding capacityMaximumCommentsoperating flowHiTrap Benzamidine FF Trypsin,35 mg/column4 ml/min(1 ml column)Prepacked columns(high sub)Trypsin,175 mg/column15 ml/min(5 ml column)Ligand density CompositionpH stabilityMean particle sizeBenzamidine Sepharose Trypsin,35 mg/ml medium300 cm/hSupplied as a4 Fast Flow(high sub)suspension ready for column packingBenzamidine 12 molesAmide coupling of ligandShort term 1990 mSepharose 4 p-aminobenzamidine/mlvia a 14 atom spacer toLong term 28Fast Flowhighly cross-linked 4%(high sub)agaroseBenzamidine SepharoseONNHNH2ONOHOHOHHSepharose(partial structure)小配体亲和填料00.200.400.600.801.003.02.52.01.51.00.5AAA2802804050.05.010.015.0mlIU/litre Activity presented as proteolytic activity/mg proteinRemoval of serine proteases from human plasma,on HiTrap Benzamidine FF(high sub),1 ml10-3 x小配体亲和填料Benzamidine Sepharose柱上GST 融合蛋白酶切及其 thrombin酶的去除000.100.200.200.30Thrombin activity0.400.800.600fr.6Cleaved SH2 proteinThrombinThrombinHigh saltbuffer washElution of HiTrapBenzamidine FF(high sub)Elution ofGSTrap FFGST-tagfr.7fr.8fr.14fr.21fr.22fr.2102050ml1525A280 nmA405 nmA)A)A)B)B)A)A)B)GSTrap FF,1 ml HiTrap Benzamidine FF(high sub),1 mlSH2-GST fusion protein in E.coli homogenate,on GSTrap FF and HiTrap Benzamidine FF(high sub),1 mlMr97 00066 00045 00030 00020 10014 400123456789SDS PAGE小配体亲和填料Binding capacity/mlMaximumCommentsmediumoperating flowLigand CompositionpH stabilityMean particle density sizeArginine Sepharose 4BNo data available75 cm/hSupplied as a ready suspensionfor column packing.Arginine Sepharose 4B1420 Arginine is coupled by an epoxy Short term 21390 m moles/ml coupling method through a Long term 213 long hydrophilic spacer and stable ether and alkylamine bonds.4OH2222222NHNHOOOCHCHCHCHCH2CH2CH_NHNH CCHCHCOOCHOH(CH)Sepharose(partial structure)小配体亲和填料Arginine SepharoseBinding capacityMaximumCommentsoperating flowHiTrapBovine antithrombin III,3 mg/column4 ml/min(1 ml column)Prepacked columns.Heparin HPBovine antithrombin III,15 mg/column20 ml/min(5 ml column)HiPrep 16/10Bovine antithrombin III,40 mg/column10 ml/minPrepacked 20 ml column.Heparin FFHeparinBovine antithrombin III,2 mg/ml medium400 cm/h*Supplied as a suspensionSepharose ready for column packing.6 Fast FlowLigandCompositionpH stabilityMean particle density sizeHiTrap Heparin HP10 mg/mlHeparin coupled to Short term 51034 mSepharose High Performance Long term 510by reductive amination to give astable attachment even in alkaline conditions.Heparin Sepharose5 mg/mlHeparin coupled to Sepharose 6Short term 41390 m6 Fast FlowFast Flow by reductive aminationLong term 412to give a stable attachment evenHiPrep 16/10in alkaline conditions.Heparin FF小配体亲和填料Heparin SepharoseHexuronic acid can either be D-glucuronic acid(top)or the C-5 epimer,L-iduronic acid(bottom).R1=-H or-SO3,R2=-SO3 or-COCH3.(A)(B)OOOCOO OHOHOOCOO OHOR1OOOOH2COR1OHHNR2Heparin Sepharose小配体亲和填料Partial purification of recombinant DNA binding Oct-1 protein Courtesy of Dr Gunnar Westin,University Hospital,Uppsala,SwedenA280nm1.00.50.0Pool0.020.040.060.080.0V olume(ml)Flow throughEluateMr97 00066 00045 00030 00020 10014 40012344321Mr97 00066 00045 00030 00020 10014 400SDS PAGEWestern blotLane 1.Starting materialLane 2.Flow-throughLane 3.LMW Calibration Kit Lane 4.Eluate poolLane 1.Starting materialLane 2.Flow-throughLane 3.LMW Calibration KitLane 4.Eluate poolBinding buffer:20 mM Tris-HCl,5%(v/v)glycerol,0.1 mM EDTA,10 mM 2-mercaptoethanol,0.1 mM Pefabloc,0.5 M NaCl,pH 8Elution buffer:Binding buffer+2 M NaCl,pH 8Column:HiTrap Heparin HP,5 ml小配体亲和填料Heparin SepharosePurification of coagulation factorsLane 1.LMW Calibration KitLane 2.Peak IILane 3.Peak ILane 4.Unbound materialA280 nmA2800.80100.60.40.250 60 70 80 90 100110 120130 ml50100%Elution bufferFlow throughpeak Ipeak II12 34Mr97 00066 00045 00030 00020 10014 400Antithrombin III from bovine plasma,on HiTrap Heparin HP,1 mlBinding buffer:0.1 M Tris-HCl,0.01 M citric acid,0.225 M NaCl,pH 7.4Elution buffer:0.1 M Tris-HCl,0.01 M citric acid,2.0 M NaCl,pH 7.4SDS PAGE,reduced小配体亲和填料Heparin SepharoseBinding capacity/mlMaximumCommentsmediumoperating flowLigand density ompositionpH stability Mean particle sizeGelatin Sepharose 4B1 mg human plasma fibronectin75 cm/hSupplied as a suspension ready for column packing.Gelatin4.58 mg gelatin/mlGelatin linked to Sepharose Short term 31090 mSepharose 4Busing the CNBr methodLong term 310小配体亲和填料Gelatin Sepharose抗体亲和填料抗体亲和填料主要是Protein A于Protein G。填料类型：rProtein A Sepharose:基因工程重组的蛋白A,通过巯基偶联,具有方向性,载量较高。nrProtein A Sehparose:无任何动物成分来源的基因工程重组蛋白A等,使用溴化氢偶联,脱落较少,无方向性,载量稍低。rmpProtein A Sepharose:蛋白A通过多位点螯合到基架上,极低脱落。MabSelect 家族：可以耐受0.1-0.5M NaOH进行CIP，节省清洗成本，延长使用寿命。（详细信息请参考？）颜料亲和填料主要用于纯化白蛋白、干扰素、凝血因子等。填料类型：Blue Sepharose:该类填料的配基模拟天然活性物质的结构，从而达到纯化目的，单是不是精确模拟。（详细填料信息请参考凝胶选择指南）Binding capacityMaximumCommentsoperating flowHiTrap Blue HPHuman serum albumin,20 mg/column4 ml/min(1 ml column)Prepacked columns.Human serum albumin,100 mg/column20 ml/min(5 ml column)Blue SepharoseHuman serum albumin,18 mg/ml medium750 cm/hSupplied as a6 Fast Flow*suspension ready for column packing.Ligand andCompositionpH stability Mean particle density sizeHiTrap Blue HPCibacron Blue F3G-ALigand coupled toShort term 31334 m4 mg/mlSepharose HighLong term 412Performance usingthe triazine method.BlueCibacron Blue F3G-ALigand coupled toShort term 31390 mSepharose 66.77.9 moles/mlSepharose Fast Flow Long term 412Fast Flowusing the triazine method.颜料亲和填料Blue SepharoseSepharoseNNNHSO3NaSO3NaNH2SO3NaNHOOONHN(partial structure)颜料亲和填料Blue SepharosePurification of human serum albumin and interferon b 000Elution buffer 1Elution buffer 2510Volume(ml)A280nmIFN-activityIFN-act.units0.02500.041000.061500.082000.102500.12A280 nmColumn:Blue Sepharose 6 Fast Flow(0.5 ml)Binding buffer:0.02 M phosphate,0.15 M NaCl,pH 7.2Elution buffer 1:0.02 M phosphate,2 M NaCl,pH 7.2Elution buffer 2:0.02 M phosphate,2 M NaCl,50%ethylene glycol,pH 7.2颜料亲和填料Blue Sepharose外源凝集素亲和填料固相外源凝集素能和数种糖类残基发生可逆反应。适合纯化多糖和糖蛋白，以及凝集红血球和肿瘤细胞。填料类型：ConA Sepharose:纯化多糖和糖蛋白Lentil Lectin Sepharose：凝集红血球和肿瘤细胞（详细填料信息请参考凝胶选择指南）LectinSpecificityMannose/glucose binding lectinsCon A,Canavalia ensiformisBranched mannoses,carbohydrates with terminal mannose or glucose(aMan aGlc GlcNAc).Lentil Lectin,Lens culinarisBranched mannoses with fucose linked a(1,6)toN-acetyl-glucosamine,(aMan aGlc GlcNAc).N-acetylglucosamine binding lectinsWheat Germ Lectin,Triticum vulgareChitobiose core of N-linked oligosaccharides,GlcNAc(b1,4GlcNAc)12 bGlcNac.外源凝集素亲和填料Con A Sepharose4BBinding capacity/mlMaximumCommentsmediumoperating flowCon A Sepharose 4B Porcine thyroglobulin,75 cm/hSupplied as a suspension 2045 mg ready for column packing.Binding capacity/mlMaximumCommentsmediumoperating flowLentil Lectin Sepharose 4B Porcine thyroglobulin,75 cm/hSupplied as a suspension 1635 mg ready for column packing.Binding capacity/mlMaximumCommentsmediumoperating flowAgarose Wheat Germ LectinNo data available75 cm/hSupplied as a suspension ready for column packing.Lentil Lectin Sepharose 4BAgarose Wheat Germ Lectin外源凝集素亲和填料Ligand CompositionpH stability*Mean particle density sizeCon A