Ion Exchange Chromatography.ppt

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1、Ion Exchange ChromatographyUseful at all stages of purification and at all scales1Scope of this presentationSome typical applicationsMechanism and principlesPractical aspects2What is ion exchange chromatography?Ion exchange chromatography is a form of adsorption chromatography which separates molecu

2、les on the basis of their charge.3Why use ion exchange?Useful at all stages of purification and at all scalesControllableHigh selectivityHigh capacityConcentratingHigh recovery4CaptureCapture biomolecules directly from clarified feed-stocks for effective initial purification1 2 3 4 5 6 7Lane 1:-Lane

3、 2:LMW markersLane 3:Starting materialLane 4:Flow throughLane 5:1st peak(containing a-amylase)Lane 6:2nd peakLane 7:LMW markersQ Sepharose XL:rec a-amylase,E.coli5ConcentrationSTREAMLINE DEAE:rec a-amylase,E.coli6Versatility:DNA Binding Protein PurificationTechniqueDNA-1 Sepharose CIEXACACACCIEXPuri

4、ficationfactorComment584943 General AC step for DNAbinding proteins Removal step,non-specificDNA binding activity removed Main purification step Final polishing,20 mg proteinobtainedJ.Berthelsen et al.(1996)J.Biol.Chem.271,3822-383092447 Rapid captureHeLa cell nuclearextractsSP Sepharose High Perfor

5、manceHeparin Sepharose Fast FlowDNA-2 Sepharose Mono S7 Final polishing and purity check,20 mg obtainedVersatility:Membrane Protein PurificationTechniqueACAIEXCIEXACCIEXPurificationfactorComment341442 Negative step;contaminant removed Detergent exchange,volume reduction before AC Main purification s

6、tepT.White et al.(1995)J.Biol.Chem.270,24156-241656242 Step gradient,rapid concentratingcapture stepPlacenta extract in1.5%Triton X-100Blue SepharoseDEAE SephacelSP Sepharose FFMuc2 SepharoseMono S8Controllable separationResults can be optimised via several factorsEffect of pH on fractionation of mo

7、del proteins9Small scaleMini Q PC 3.2/3:Glutathione synthetaseSDS-PAGEStarting materialPeak 210What happens in ion exchange?Sampleapplicationand washElutionEquilibrationRegeneration-+-11What happens in ion exchange?Equilibration+-anion exchange bead12What happens in ion exchange?Sample applicationan

8、d wash-+-13What happens in ion exchange?Elution-+-14What happens in ion exchange?Regeneration-+15Separation by chargeInteraction between opposite chargesCharged groups on the proteins interact with charged groups on the ion exchanger.Different proteins have different charges and interact differently

9、.Anion or cation exchangeWhen the protein is negatively charged,it is an anion-anion exchangeWhen it is positively charged,it is a cation-cation exchange16Basis for selectivitySome of the charged regions which will influence ion exchangeDifferent proteins have different charges and different pattern

10、s of surface charge17Effect of pH on chargeNH3RCOOH+NH3RCOO+-RNH2COO-Low pHPositive chargeHigh pHNegative chargeHydrogen gainedHydrogen lost18Titration curvesOverall charge on protein-+NH3RCOOH+NH3RCOO+-RNH2COO-acid isoelectric point alkalineexcess positive charge balanced positive and negative char

11、ge excess negative chargeThe overall charge on a protein depends on pHpH31019Controlling selectivity by pHCharge on protein-+pH310Anion exchangerCation exchanger20Practical ConsiderationsWhat equipment do we need?What ion exchanger should we use?How are we going to run the separation?21pHUVCondFract

12、ion collection FRAC-900AMIXERW1W2BSamplePump P-920Valve FV-903Monitor UPC-900Different buffersInjection Valve V1Large and small fractionsA versatile set-up for ion exchangeColumns for AIX,CIX and buffer exchangeMonitor for UV,conductivity and pH22The right ion exchangerThere is no single perfect ion

13、 exchanger!Different ion exchangers are needed for different sample loads and different purposes23Functional properties of ion exchangersCharged groupsTypeDensityMatrixPorosityParticle sizeChemistrySelectivityCapacityCapacity,speedPeak width,speedUseful life,recovery24Charged groupsAnion exchangers-

14、Diethylaminoethyl(DEAE)-OCH2CH2N+(CH2CH3)2-Quaternary aminoethyl(QAE)-OCH2CH2N+(C2H5)2CH2CHOHCH3-Quaternary ammonium(Q)-CH2N+(CH3)3Cation exchangers-Carboxymethyl(CM)-OCH2COO-Sulphopropyl(SP)-CH2CH2CH2SO3-Methylsulphonate(S)-CH2SO325Strong and weak ion exchangersWeak ion exchangers:capacity varies w

15、ith pHStrong ion exchangers:capacity is constant over a wide range of pH Weak anionexchangerWeak cationexchangerStrong anion exchangerStrong cation exchanger26Advantages of strong ion exchangersCharged at all pHs we want to useConstant charge at all pHs we want to useFaster and easier to equilibrate

16、27Matrix and speedMechanically strong beads work at high flow ratesSmall beads for fast kineticsBig beads for high volume flow ratesX28Matrix and resolution3 mm Mini S10 mm Mono S29Porosity is important for capacity30Modern matricesPhysically strong for fast resultsHigh capacitiesVery reproducibleDi

17、fferent sizes for different scales of operation31Types of capacityTotal ionic capacityAvailable capacityDynamic capacitye.g.3.5 mM/mLnot important in the labe.g.25 mg HSA/mLvaries with pH,sample,ionic strength e.g 25 mg HSA/mL,300 cm/hflow rate dependentalso varies with pH,sample,ionic strength32Med

18、iaPlease read the notes!33Batch-to-batch reproducibilityQC evaluation of 4 batches of SOURCE 30S34Monodisperse mediaMiniBeadsMonoBeadsSOURCE 15SOURCE 303 mm10 mm15 mm30 mmmicro-purificationpolishingpolishingintermediate steps35Which ion exchangers?Capture from crude sampleIntermediate purificationHi

19、gh performance polishingHiPrep 16/10 Q&SP XLMono Q&Mono S HR 5/5Lab scale purification of 1-10 mg protein36Column sizeVolume to suit amount(mass)of proteinUse 5-40%of available capacityLength 5-10 cmIon exchange columnShort,fat37The Right ConditionspHAdditivesElution schemesFlow rate38Selecting buff

20、er systemAnion exchangeCationic buffering ions,pH slightly above the pH where the target protein is boundHigher pH will increase the tightness of binding Cation exchangeAnionic buffering ions,pH slightly below the value where the target protein is boundLower pH will increase the tightness of binding

21、39Buffers for anion exchangepH456789101112N-MethylpiperazinePiperazineL-HistidineBis-TrisBis-Tris-propaneTriethanolamineTrisN-methyldiethane1,3-diaminopropaneEthanolamine(hi)Piperazine(hi)1,3-diaminoPiperidineBufferPrep AIEX40Buffers for cation exchange411)Put 1 ml ion exchanger into several tubes 2

22、)Add buffers with different pHs3)Add sample,mix4)Analyse the supernatants for the protein of interestHere the target binds completely at pH 7.5 and above.Conclusion:Anion exchanger,initial pH 7.5.Test tube method to determine the initial pHpH 6.0 6.5 7.0 7.5 8.042Determining optimal pH by scoutingmA

23、UElution volume(ml)M NaCl10.50pH 5pH 6pH 7pH 8pH 9pH scouting for the separation of pancreatinConditions:System:KTAexplorer 100,BufferPrepColumn:RESOURCE Q,6 mlSample:2 mg crude pancreatin43Practical tips for buffersBuffer with same charge as ion exchangerAdjust pH with same ion as in the saltAdjust

24、 pH after salt additionAdjust pH at the working temperatureMonitor pH and conductivity during the run Blank run!442050 mM buffer salt is usually enough The pKa of the buffer should not differ by more than 0.5 pH units from the working pHTo ensure the proper pH value,dissolve the sample in buffer A,c

25、ritical with large samplesMore buffer tips45A:Citrate 20 mM,pH 4.9B:Citrate 20 mM,NaCl 1 M,pH 4.9A:Citrate 20 mM,pH 4.9 B:A+NaCl 1 M Different ways of making the same buffer46 To ensure a proper pH value,the sample should be dissolved in buffer A,(critical with large volume samples).Buffering ion co

26、ncentrations of 2050 mM are usually sufficient.AUvolumepH valuegradientpH value and buffer capacityvolumeAUpH valuegradient47What if the buffering ions bind to the ion exchanger?When A-binds the equilibrium is disturbed,and the pH changesAs well as that you lose buffer capacity!48If the buffering io

27、n does bind If you must use a buffer like this:Make sure the buffer concentration is high enoughMake sure the column is well equilibrated and saturated with bound buffering ions.Elute with a gradient of buffer concentration49Eluent additivesUn-charged,neutral additives are usually OKUse only positiv

28、ely charged additives with anion exchangers.and negatively charged ones with cation exchangers50Additives and potential problemsAccumulation and elution of EDTA in anion exchange500100%BTime(min)RNA/DNA02468RNADNAEDTA absorbs at 254 nm51Additives and potential problemsDetergents which bind to the co

29、lumnAnionic detergents(e.g.SDS)will bind to an anion exchangerCationic detergents(e.g.CPC)will bind to an cation exchangerIt is a waste of time trying to remove them!so make sure you dont use them the wrong way round52Above the critical micelle concentration(CMC),detergents form micelles which cause

30、 problems with UV detection.Additives and potential problemsDetergents reaching their CMC in a salt gradient53Some words of cautionCheck the solubility and stability of the sample moleculesOrganic solvents may increase the pKa of the bufferWatch out for increases in viscosity and back-pressure due t

31、o glycerol and other alcoholsRun blanks when using detergents54Elution schemesIsocraticStep gradientLinear gradientAdaptedpH gradientSalt gradientBoth pH and saltNot usualExcellent for capture stepsStandard methodSaves time and improves resultsSeldom usedStandard methodCan be tricky!55Note different

32、 salt concentrations for similar elution strength:Sulphate(SO4-2)150 mM Chloride(Cl-1)350 mM Acetate(CH3COO-1)700 mMChanging the eluting salt corresponds,in most cases,to changing the gradient slope(differences in elution strength)Salt gradients56Step gradient for captureHiPrep 16/10 Q XL:rec DAOCS,

33、E.coliLinear gradient,starting pointOptimised step elution57Gradient step splits a single peakStep gradient artifact!HiPrep 16/10 Q XL:rec DAOCS,E.coli58Linear gradients for fractionationStandard conditions for first tryLonger gradients improve resolutionBuffer A:20-50 mMBuffer B:20-50 mM+1 M NaCl05

34、0%B in 20 col vol51100%in 35 col vol100-0%in 3-5 col volSeparationWashRe-equilibrate59pH gradientsDecreasing pH for anion exchangersTitrates COO-groups on the proteinsIncreasing pH for cation exchangersTitrates NH3+groups on the proteins60Optimising flow rateUse high flow rate for:High sample throug

35、hput High productivityLower the flow rate for:Maximum resolution1800 cm/h(4.98 ml/min)900 cm/h(2.49 ml/min)300 cm/h(0.83 ml/min)200 cm/h(0.55 ml/min)040Time(min)0270 904.561Preparing the sampleFilter or centrifuge to remove particles which can clog columnsAdjust the pH and salt concentration by buff

36、er exchange on Sephadex G-25 or by dilutionLarge sample volumes are OK if pH and salt concentration are OK62Re-equilibrationAfter elutionRemove tightly bound components with 1 M saltRe-equilibrate with ca 5 col vol start buffer63Cleaning columnsMost modern ion exchangers can be cleaned with 1 M NaOH-removes most things!Wash well with water and re-equilibrateNeutral detergents and enzymes can also be used,but can be difficult to get rid of afterwards64SummaryUseful at all stages of purification and at all scalesControllableHigh selectivityHigh capacityConcentratingHigh recovery65