1、Ixv_2 JB 980910,1,Ion Exchange Chromatography,Useful at all stages of purification and at all scales,Ixv_2 JB 980910,2,Scope of this presentation,Some typical applications Mechanism and principles Practical aspects,Ixv_2 JB 980910,3,What is ion exchange chromatography?,Ion exchange chromatography is
2、 a form of adsorption chromatography which separates molecules on the basis of their charge.,Ixv_2 JB 980910,4,Why use ion exchange?,Useful at all stages of purification and at all scales Controllable High selectivity High capacity Concentrating High recovery,Ixv_2 JB 980910,5,Capture, Capture biomo
3、lecules directly from clarified feed-stocks for effective initial purification,1,2,3,4,5,6,7,Lane 1: - Lane 2: LMW markers Lane 3: Starting material Lane 4: Flow through Lane 5: 1st peak (containing a-amylase) Lane 6: 2nd peak Lane 7: LMW markers,Q Sepharose XL: rec a-amylase, E. coli,Ixv_2 JB 98091
4、0,6,Concentration,STREAMLINE DEAE: rec a-amylase, E. coli,Ixv_2 JB 980910,7,Versatility: DNA Binding Protein Purification,Technique,DNA-1 Sepharose,CIEX,AC,AC,AC,CIEX,Purification factor,Comment,5,8,4943,General AC step for DNA binding proteins,Removal step, non-specific DNA binding activity removed
5、,Main purification step,Final polishing, 20 mg protein obtained,J. Berthelsen et al. (1996) J. Biol. Chem. 271, 3822-3830,9,2447,Rapid capture,HeLa cell nuclear extracts,SP Sepharose High Performance,Heparin Sepharose Fast Flow,DNA-2 Sepharose,Mono S,Ixv_2 JB 980910,8,Final polishing and purity chec
6、k, 20 mg obtained,Versatility: Membrane Protein Purification,Technique,AC,AIEX,CIEX,AC,CIEX,Purification factor,Comment,3,4,1442,Negative step; contaminant removed,Detergent exchange, volume reduction before AC,Main purification step,T. White et al. (1995) J. Biol. Chem. 270, 24156-24165,6,242,Step
7、gradient, rapid concentrating capture step,Ixv_2 JB 980910,9,Controllable separation,Results can be optimised via several factors,Effect of pH on fractionation of model proteins,Ixv_2 JB 980910,10,Small scale,Mini Q PC 3.2/3: Glutathione synthetase,SDS-PAGE,Starting material,Peak 2,Ixv_2 JB 980910,1
8、1,What happens in ion exchange?,Sample application and wash,Elution,Equilibration,Regeneration,-,-,-,-,-,-,-,-,-,-,-,-,-,-,Ixv_2 JB 980910,12,What happens in ion exchange?,Equilibration,+,+,+,+,+,+,-,-,-,-,-,-,-,anion exchange bead,Ixv_2 JB 980910,13,What happens in ion exchange?,Sample application
9、and wash,-,+,+,+,+,+,+,-,-,-,-,Ixv_2 JB 980910,14,What happens in ion exchange?,Elution,-,-,+,+,+,+,+,+,+,+,+,+,+,+,-,-,Ixv_2 JB 980910,15,What happens in ion exchange?,Regeneration,+,+,+,+,+,Ixv_2 JB 980910,16,Separation by charge,Interaction between opposite charges Charged groups on the proteins
10、interact with charged groups on the ion exchanger. Different proteins have different charges and interact differently. Anion or cation exchange When the protein is negatively charged, it is an anion - anion exchange When it is positively charged, it is a cation - cation exchange,Ixv_2 JB 980910,17,B
11、asis for selectivity,Some of the charged regions which will influence ion exchangeDifferent proteins have different charges and different patterns of surface charge,Ixv_2 JB 980910,18,Effect of pH on charge,NH3,R,COOH,+,NH3,R,COO,+,-,R,NH2,COO,-,Low pHPositive charge,High pHNegative charge,Hydrogen
12、gained,Hydrogen lost,Ixv_2 JB 980910,19,Titration curves,Overall charge on protein,-,+,acid isoelectric point alkaline excess positive charge balanced positive and negative charge excess negative charge,The overall charge on a protein depends on pH,pH,3,10,Ixv_2 JB 980910,20,Controlling selectivity
13、by pH,pH,3,10,Anion exchanger,Cation exchanger,Ixv_2 JB 980910,21,Practical Considerations,What equipment do we need?What ion exchanger should we use?How are we going to run the separation?,Ixv_2 JB 980910,22,Fraction collection FRAC-900,A,MIXER,W1,W2,B,Sample,Pump P-920,Valve FV-903,Monitor UPC-900
14、,Different buffers,Injection Valve V1,Large and small fractions,A versatile set-up for ion exchange,Columns for AIX, CIX and buffer exchange,Monitor for UV, conductivity and pH,Ixv_2 JB 980910,23,The right ion exchanger,There is no single perfect ion exchanger!Different ion exchangers are needed for
15、 different sample loads and different purposes,Ixv_2 JB 980910,24,Functional properties of ion exchangers,Charged groups Type DensityMatrix Porosity Particle size Chemistry,Selectivity CapacityCapacity, speed Peak width, speed Useful life, recovery,Ixv_2 JB 980910,25,Charged groups,Anion exchangers
16、-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) -CH2SO3,Ixv_2 JB 980910,26,Strong and weak ion exchanger
17、s,Weak ion exchangers: capacity varies with pHStrong ion exchangers: capacity is constant over a wide range of pH,Weak anion exchanger,Weak cation exchanger,Strong anion exchanger,Strong cation exchanger,Ixv_2 JB 980910,27,Advantages of strong ion exchangers,Charged at all pHs we want to useConstant
18、 charge at all pHs we want to useFaster and easier to equilibrate,Ixv_2 JB 980910,28,Matrix and speed,Mechanically strong beads work at high flow ratesSmall beads for fast kinetics Big beads for high volume flow rates,Ixv_2 JB 980910,29,Matrix and resolution,3 mm Mini S,10 mm Mono S,Ixv_2 JB 980910,
19、30,Porosity is important for capacity,Ixv_2 JB 980910,31,Modern matrices,Physically strong for fast results High capacities Very reproducible Different sizes for different scales of operation,Ixv_2 JB 980910,32,Types of capacity,Total ionic capacityAvailable capacityDynamic capacity,e.g. 3.5 mM/mL n
20、ot important in the lab e.g. 25 mg HSA/mL varies with pH, sample, ionic strength e.g 25 mg HSA/mL, 300 cm/h flow rate dependent also varies with pH, sample, ionic strength,Ixv_2 JB 980910,33,Media,Please read the notes!,Ixv_2 JB 980910,34,Batch-to-batch reproducibility,QC evaluation of 4 batches of
21、SOURCE 30S,Ixv_2 JB 980910,35,Monodisperse media,MiniBeads MonoBeads SOURCE 15 SOURCE 30,3 mm 10 mm 15 mm 30 mm,micro-purification polishing polishing intermediate steps,Ixv_2 JB 980910,36,Which ion exchangers?,Capture from crude sample Intermediate purification High performance polishing,HiPrep 16/
22、10 Q & SP XL Mono Q & Mono S HR 5/5,Lab scale purification of 1 - 10 mg protein,Ixv_2 JB 980910,37,Column size,Volume to suit amount (mass) of protein Use 5-40% of available capacityLength 5-10 cm,Ion exchange column,Short, fat,Ixv_2 JB 980910,38,The Right Conditions,pH Additives Elution schemes Flo
23、w rate,Ixv_2 JB 980910,39,Selecting buffer system,Anion exchange Cationic buffering ions, pH slightly above the pH where the target protein is bound Higher pH will increase the tightness of binding Cation exchange Anionic buffering ions, pH slightly below the value where the target protein is bound
24、Lower pH will increase the tightness of binding,Ixv_2 JB 980910,40,Buffers for anion exchange,pH,4,5,6,7,8,9,10,11,12,N-Methylpiperazine,Piperazine,L-Histidine,Bis-Tris,Bis-Tris-propane,Triethanolamine,Tris,N-methyldiethane,1,3-diaminopropane,Ethanolamine,(hi) Piperazine,(hi)1,3-diamino,Piperidine,B
25、ufferPrep AIEX,Ixv_2 JB 980910,41,Buffers for cation exchange,Ixv_2 JB 980910,42,1) Put 1 ml ion exchanger into several tubes 2) Add buffers with different pHs 3) Add sample, mix 4) Analyse the supernatants for the protein of interestHere the target binds completely at pH 7.5 and above. Conclusion:
26、Anion exchanger, initial pH 7.5.,Test tube method to determine the initial pH,pH 6.0 6.5 7.0 7.5 8.0,Ixv_2 JB 980910,43,Determining optimal pH by scouting,pH scouting for the separation of pancreatinConditions: System: KTAexplorer 100, BufferPrep Column: RESOURCE Q, 6 ml Sample: 2 mg crude pancreati
27、n,Ixv_2 JB 980910,44,Practical tips for buffers,Buffer with same charge as ion exchanger Adjust pH with same ion as in the salt Adjust pH after salt addition Adjust pH at the working temperature Monitor pH and conductivity during the run Blank run!,Ixv_2 JB 980910,45,2050 mM buffer salt is usually e
28、nough The pKa of the buffer should not differ by more than 0.5 pH units from the working pH To ensure the proper pH value, dissolve the sample in buffer A, critical with large samples,More buffer tips,Ixv_2 JB 980910,46,A: Citrate 20 mM, pH 4.9 B: Citrate 20 mM, NaCl 1 M, pH 4.9,A: Citrate 20 mM, pH
29、 4.9 B: A + NaCl 1 M,Different ways of making the same buffer,Ixv_2 JB 980910,47,To ensure a proper pH value, the sample should be dissolved in buffer A, (critical with large volume samples).Buffering ion concentrations of 2050 mM are usually sufficient.,AU,volume,pH value,gradient,pH value and buff
30、er capacity,volume,AU,pH value,gradient,Ixv_2 JB 980910,48,What if the buffering ions bind to the ion exchanger?,When A- binds the equilibrium is disturbed, and the pH changes As well as that you lose buffer capacity!,Ixv_2 JB 980910,49,If the buffering ion does bind,If you must use a buffer like th
31、is: Make sure the buffer concentration is high enough Make sure the column is well equilibrated and saturated with bound buffering ions. Elute with a gradient of buffer concentration,Ixv_2 JB 980910,50,Eluent additives,Un-charged, neutral additives are usually OK Use only positively charged additive
32、s with anion exchangers . and negatively charged ones with cation exchangers,Ixv_2 JB 980910,51,Additives and potential problems,Accumulation and elution of EDTA in anion exchange,EDTA absorbs at 254 nm,Ixv_2 JB 980910,52,Additives and potential problems,Detergents which bind to the column,Anionic d
33、etergents (e.g. SDS) will bind to an anion exchanger Cationic 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 round,Ixv_2 JB 980910,53,Above the critical micelle concentration (CMC), detergents form micell
34、es which cause problems with UV detection.,Additives and potential problems,Detergents reaching their CMC in a salt gradient,Ixv_2 JB 980910,54,Some words of caution,Check the solubility and stability of the sample molecules Organic solvents may increase the pKa of the buffer Watch out for increases
35、 in viscosity and back-pressure due to glycerol and other alcohols Run blanks when using detergents,Ixv_2 JB 980910,55,Elution schemes,Isocratic Step gradient Linear gradient AdaptedpH gradient Salt gradient Both pH and salt,Not usual Excellent for capture steps Standard method Saves time and improv
36、es resultsSeldom used Standard method Can be tricky!,Ixv_2 JB 980910,56,Note different salt concentrations for similar elution strength:Sulphate (SO4-2) 150 mM Chloride (Cl-1) 350 mMAcetate (CH3COO-1) 700 mM Changing the eluting salt corresponds, in most cases, to changing the gradient slope (differ
37、ences in elution strength),Salt gradients,Ixv_2 JB 980910,57,Step gradient for capture,HiPrep 16/10 Q XL: rec DAOCS, E. coli,Linear gradient, starting point,Optimised step elution,Ixv_2 JB 980910,58,Gradient step splits a single peak,Step gradient artifact!,HiPrep 16/10 Q XL: rec DAOCS, E. coli,Ixv_
38、2 JB 980910,59,Linear gradients for fractionation,Standard conditions for first try Longer gradients improve resolutionBuffer A: 20-50 mM Buffer B: 20-50 mM + 1 M NaCl050 % B in 20 col vol 51100% in 35 col vol100-0% in 3-5 col vol,Separation,Wash,Re-equilibrate,Ixv_2 JB 980910,60,pH gradients,Decrea
39、sing pH for anion exchangers Titrates COO- groups on the proteinsIncreasing pH for cation exchangers Titrates NH3+ groups on the proteins,Ixv_2 JB 980910,61,Optimising flow rate,Use high flow rate for:High sample throughputHigh productivityLower the flow rate for:Maximum resolution,Ixv_2 JB 980910,6
40、2,Preparing the sample,Filter or centrifuge to remove particles which can clog columns Adjust the pH and salt concentration by buffer exchange on Sephadex G-25 or by dilution Large sample volumes are OK if pH and salt concentration are OK,Ixv_2 JB 980910,63,Re-equilibration,After elution Remove tigh
41、tly bound components with 1 M saltRe-equilibrate with ca 5 col vol start buffer,Ixv_2 JB 980910,64,Cleaning columns,Most 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 afterwards,Ixv_2 JB 980910,65,Summary,Useful at all stages of purification and at all scales Controllable High selectivity High capacity Concentrating High recovery,
