Figure 4 shows the simple procedure used to perform affinity purification on prepacked HiTrap columns. olumn 5-5 Fig.4. HiTrap columns may be used with a syringe,a peristaltic p mp or a liquid chromatography system (see Selection of Purification Equi pplied witha detailed protocol to ensure optimum res Media selection A ligand already coupled to a matrix is the simplest solution.Selecting prepacked columns such as HiTrap or HiPrep will not only be more convenient,but will also save time in method optimization as these columns are supplied with detailed instructions for optimum performance If a ligand is available,but needs to be coupled to a pre-activated matrix,refer to Chapter 5. abe.dide whtherr orh the time and effor ipvolved o ity mediu m.In many cases,it may interaction chromatography. Preparation of media and buffers rvatives should be 兰 shed away oughly before using an R nity mec dia supplied as freeze-dried powders in the correct Use high quality wate and chemicals.Solutions should be filtered through 0.45 m or 0.22 um filters 业 Reuse of affinity media dep ends on the nature of the sample and should only be considered 业 If an affinity medium is to be used routinely,care must be taken to ensure that any contaminants from the crude sample can be removed by procedures that do not damage the ligand. Binding and elution buffers are specific for each affinity medium since it is their influence on the interaction between the target molecule and the ligand that facilitates the affinity- based separation.Some affinity media may also require a specific buffer in order to make the medium ready for use again
16 Figure 4 shows the simple procedure used to perform affinity purification on prepacked HiTrap columns. Equilibrate column with binding buffer Apply sample Wash with binding buffer Waste Collect Elute with elution buffer Collect fractions 3 min 5-15 min 2 min Fig. 4. HiTrap columns may be used with a syringe, a peristaltic pump or a liquid chromatography system (see Selection of Purification Equipment, Appendix 2) and are supplied with a detailed protocol to ensure optimum results. Media selection A ligand already coupled to a matrix is the simplest solution. Selecting prepacked columns such as HiTrap or HiPrep will not only be more convenient, but will also save time in method optimization as these columns are supplied with detailed instructions for optimum performance. If a ligand is available, but needs to be coupled to a pre-activated matrix, refer to Chapter 5. If no suitable ligand is available, decide whether it is worth the time and effort involved to obtain a ligand and to develop a specific affinity medium. In many cases, it may be more convenient to use alternative purification techniques such as ion exchange or hydrophobic interaction chromatography. Preparation of media and buffers Storage solutions and preservatives should be washed away thoroughly before using any affinity medium. Re-swell affinity media supplied as freeze-dried powders in the correct buffer as recommended by the manufacturer. Use high quality water and chemicals. Solutions should be filtered through 0.45 µm or 0.22 µm filters. Reuse of affinity media depends on the nature of the sample and should only be considered when processing identical samples to avoid cross-contamination. If an affinity medium is to be used routinely, care must be taken to ensure that any contaminants from the crude sample can be removed by procedures that do not damage the ligand. Binding and elution buffers are specific for each affinity medium since it is their influence on the interaction between the target molecule and the ligand that facilitates the affinitybased separation. Some affinity media may also require a specific buffer in order to make the medium ready for use again
they may damage the rotation end-over-end stirring. Sample preparation and application Samples should be clear and free from particulate matter.Simple steps to clarify a sample before beginning purification will avoid clogging the column,may reduce the need for stringent washing procedures and can extend the life of the chromatographic medium. Appendix 1 contains an overview of sample preparation techniques If possible,test the affinity of the ligand:target molecule interaction.Too low affinity will ash thro ugh or leak from the To ult in lov since the target molecule may not Binding of the targe made more efficient by adjusting the sample o H of the bindin and p a buffe atein binding buffe (se aplving sample in order to allow mors timo t take interactions that are sl ow to after place before continuing to wash the column.In some cases,applying the sample in aliquots may be beneficial. s kn wn to interfere with Since affinity chromatography is a binding technique,the sample volume does not affect the separation as long as conditions are chosen to ensure that the target protein binds strongly to the ligand. It may be necessary to test for a flow rate that gives the most efficient binding during sample application since this parameter can vary according to the specific interaction between the target protein and the ligand and their concentrations. The column must be pre-equilibrated in binding buffer before beginning sample applica- tion.For interactions with strong affinity betw quickly reach equilibrium,samples can be a plied at a high flow rate.Howe erac ons with weak affinity and/or slow uilibrium.a lower flow rate should be used. The ach ould o eve effic t bind ding may vary ac g to ined hen neces ary.Further ails on the specific inetics to take place before continuing to wash the column.In some cases,applying the sample in aliquots may be beneficial. Do not begin elution of target substances until all unbound material has been washed through the column by the binding buffer(determined by UV absorbance at 280 nm).This will improve the purity of the eluted target substance
17 Avoid using magnetic stirrers as they may damage the matrix. Use mild rotation or end-over-end stirring. Sample preparation and application Samples should be clear and free from particulate matter. Simple steps to clarify a sample before beginning purification will avoid clogging the column, may reduce the need for stringent washing procedures and can extend the life of the chromatographic medium. Appendix 1 contains an overview of sample preparation techniques. If possible, test the affinity of the ligand: target molecule interaction. Too low affinity will result in poor yields since the target protein may wash through or leak from the column during sample application. Too high affinity will result in low yields since the target molecule may not dissociate from the ligand during elution. Binding of the target protein may be made more efficient by adjusting the sample to the composition and pH of the binding buffer: perform a buffer exchange using a desalting column or dilute in binding buffer (see page 133). When working with very weak affinity interactions that are slow to reach equilibrium, it may be useful to stop the flow after applying sample in order to allow more time for the interaction to take place before continuing to wash the column. In some cases, applying the sample in aliquots may be beneficial. Sample preparation techniques should ensure that components known to interfere with binding (the interaction between the target molecule and the ligand) are removed. Since affinity chromatography is a binding technique, the sample volume does not affect the separation as long as conditions are chosen to ensure that the target protein binds strongly to the ligand. It may be necessary to test for a flow rate that gives the most efficient binding during sample application since this parameter can vary according to the specific interaction between the target protein and the ligand and their concentrations. The column must be pre-equilibrated in binding buffer before beginning sample application. For interactions with strong affinity between the ligand and the target molecule that quickly reach equilibrium, samples can be applied at a high flow rate. However, for interactions with weak affinity and/or slow equilibrium, a lower flow rate should be used. The optimal flow rate to achieve efficient binding may vary according to the specific interaction and should be determined when necessary. Further details on the kinetics involved in binding and elution from affinity media are covered in Appendix 7. When working with very weak affinity interactions that are slow to reach equilibrium, it may be useful to stop the flow after applying the sample to allow more time for the interaction to take place before continuing to wash the column. In some cases, applying the sample in aliquots may be beneficial. Do not begin elution of target substances until all unbound material has been washed through the column by the binding buffer (determined by UV absorbance at 280 nm). This will improve the purity of the eluted target substance
Elution There is no generally applicable elution scheme for all affinity media.Reference to manufacturer's instructions,the scientific literature and a few simple rules should result in an effective elution method that elutes the target protein in a concentrated form.Elution methods may be either selective or non-selective,as shown in Figure 5. ired f ◆+D Fig.5.Elution msthods When substances are very tightly bound to the affinity medium,it may be useful to stop the some time afte only ed)before ntinuing elution.This give helps to improve Selective elution methods are applied in combination with group-specific ligands whereas selective methods used in ombination with hi cific ligands.Forces that mai t ex include elect ostatic intera ons,hy and n hond ing.Agents that weaken these interactions may be expected to function a efficient eluting agents. e specific e determined when necessary.Further details on the kinetics involved in binding and elution of target molecules from affinity media are covered in Appendix 7. A compromise may have to be made between the harshness of the eluent required for elution and the risk of denaturing the eluted material or damaging the ligand on the affinity medium. Ready to use affinity media from Amersham Biosciences are supplied with recommendations for the most suitable elution buffer to reverse the interaction between the ligand and target protein of the specific interaction.Each of these recommendations will be based on one of the following elution methods:
18 Elution There is no generally applicable elution scheme for all affinity media. Reference to manufacturer's instructions, the scientific literature and a few simple rules should result in an effective elution method that elutes the target protein in a concentrated form. Elution methods may be either selective or non-selective, as shown in Figure 5. Method 1 The simplest case. A change of buffer composition elutes the bound substance without harming either it or the ligand. Method 2 Extremes of pH or high concentrations of chaotropic agents are required for elution, but these may cause permanent or temporary damage. Methods 3 and 4 Specific elution by addition of a substance that competes for binding. These methods can enhance the specificity of media that use group-specific ligands. Fig. 5. Elution methods. When substances are very tightly bound to the affinity medium, it may be useful to stop the flow for some time after applying eluent (10 min. to 2 h is commonly used) before continuing elution. This gives more time for dissociation to take place and thus helps to improve recoveries of bound substances. Selective elution methods are applied in combination with group-specific ligands whereas non-selective elution methods are used in combination with highly specific ligands. Forces that maintain the complex include electrostatic interactions, hydrophobic effects and hydrogen bonding. Agents that weaken these interactions may be expected to function as efficient eluting agents. The optimal flow rate to achieve efficient elution may vary according to the specific interaction and should be determined when necessary. Further details on the kinetics involved in binding and elution of target molecules from affinity media are covered in Appendix 7. A compromise may have to be made between the harshness of the eluent required for elution and the risk of denaturing the eluted material or damaging the ligand on the affinity medium. Ready to use affinity media from Amersham Biosciences are supplied with recommendations for the most suitable elution buffer to reverse the interaction between the ligand and target protein of the specific interaction. Each of these recommendations will be based on one of the following elution methods:
pH elution A change in pH alters the degree of ionization of charged groups on the ligand and/or the bound protein.This change may affect the binding sites directly,reducing their affinity,or cause indirect changes in affinity by alterations in conformation. A step decrease in pH is the most common way to elute bound substances.The chemical stability of the matrix,ligand and target protein determines the limit of pH that may be used. If low pH must be used,collect fractions into neutralization buffer sus1M Tris-HCl pH9(60-200 ul per ml eluted fraction)to return the fraction to a neutral pH.The column should also be re-equilibrated to neutral pH immediately. lonic strength elution between the e ligand is a mild elutio Enzymes usually elute at a concentration of 1 M NaCl or less. Competitive elution Selective eluents are often used to separate substances on a group specific medium or when the binding affinity of the ligand/target protein interaction is relatively high.The eluting agent competes either for binding to the target protein or for binding to the ligand.Substances may be eluted either by a concentration gradient of a single eluent or by pulse elution, see page 22. When working with competitive elution the concentration of competing compound should be similar to the concentration of the coupled ligand.However,if the free competing compound binds more weakly than the ligand to the target molecule,use a concentration te-fodher tha igd. Reduced polarity of eluent inactivatin If other elution methods fail,deforming buffers,which alter the structure of proteins,can be used,e.g.chaotropic agents such as guanidine hydrochloride or urea.Chaotropes should be avoided whenever possible since they are likely to denature the eluted protein
19 pH elution A change in pH alters the degree of ionization of charged groups on the ligand and/or the bound protein. This change may affect the binding sites directly, reducing their affinity, or cause indirect changes in affinity by alterations in conformation. A step decrease in pH is the most common way to elute bound substances. The chemical stability of the matrix, ligand and target protein determines the limit of pH that may be used. If low pH must be used, collect fractions into neutralization buffer such as 1 M Tris-HCl, pH 9 (60–200 µl per ml eluted fraction) to return the fraction to a neutral pH. The column should also be re-equilibrated to neutral pH immediately. Ionic strength elution The exact mechanism for elution by changes in ionic strength will depend upon the specific interaction between the ligand and target protein. This is a mild elution using a buffer with increased ionic strength (usually NaCl), applied as a linear gradient or in steps. Enzymes usually elute at a concentration of 1 M NaCl or less. Competitive elution Selective eluents are often used to separate substances on a group specific medium or when the binding affinity of the ligand/target protein interaction is relatively high. The eluting agent competes either for binding to the target protein or for binding to the ligand. Substances may be eluted either by a concentration gradient of a single eluent or by pulse elution, see page 22. When working with competitive elution the concentration of competing compound should be similar to the concentration of the coupled ligand. However, if the free competing compound binds more weakly than the ligand to the target molecule, use a concentration ten-fold higher than that of the ligand. Reduced polarity of eluent Conditions are used to lower the polarity of the eluent promote elution without inactivating the eluted substances. Dioxane (up to 10%) or ethylene glycol (up to 50%) are typical of this type of eluent. Chaotropic eluents If other elution methods fail, deforming buffers, which alter the structure of proteins, can be used, e.g. chaotropic agents such as guanidine hydrochloride or urea. Chaotropes should be avoided whenever possible since they are likely to denature the eluted protein
Gradient and step elution r6howexamp of tepgradic ion conditions Por prepackd affinity Trap colu ms,rheeg elution cond ons,a step elution using a simple with a ch atography system such KTAprime The use of a chromatography system is esential when gradicnt clution s lumns can also be usec required. ig.6a.Step elution. Fig6b.Gradient clution. During development and optimization of affinity purification,use a gradient elution to scan for the optimal binding or elution conditions,as shown in Figure 7 and Figure 8. Sample: 280n 0. elution 04 05M Elution butfer: .03 02 石p5o Fig.7.Gradient elution of a(His).fusion protein. 20
20 0 0.1 0.2 0 0.1 0.2 0.3 0.4 A 280 nm 0.5 0 45 65 min Imidazole (M) 0.3 UV 280 nm Programmed elution buffer conc. (His) fusion protein6 1 2 1: selected imidazole concentration for elution of impurities 2: selected imidazole concentration for elution of pure (His) fusion protein 6 Gradient and step elution Figure 6 shows examples of step and gradient elution conditions. For prepacked affinity HiTrap columns, supplied with predefined elution conditions, a step elution using a simple syringe can be used. HiTrap columns can also be used with a chromatography system such as ÄKTAprime. The use of a chromatography system is essential when gradient elution is required. Sample: Clarified homogenate of E. coli expressing His fusion protein Column: HiTrap Chelating HP 1 ml column charged with Ni2+ Binding buffer: 20 mM sodium phosphate, 0.5 M sodium chloride, 10 mM imidazole, pH 7.4 Elution buffer: 20 mM sodium phosphate, 0.5 M sodium chloride, 0.5 M imidazole, pH 7.4 Flow: 1 ml/min System: ÄKTAprime Fig. 6a. Step elution. A280 Time/vol. Binding conditions Elution conditions A280 Time/vol. Binding conditions Linear change in elution conditions Fig. 6b. Gradient elution. During development and optimization of affinity purification, use a gradient elution to scan for the optimal binding or elution conditions, as shown in Figure 7 and Figure 8. Fig. 7. Gradient elution of a (His)6 fusion protein