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If several functional groups are available, couple the ligand via the group least likely to be involved in the specific affinity interaction. A range of pre-activated matrices for attachment of the ligand through different functional groups is available (see Table 7). Spacer arms The binding site of a target protein is often located deep within the molecule and an affinity medium prepared by coupling small ligands, such as enzyme cofactors, directly to Sepharose may exhibit low binding capacity due to steric interference i.e. the ligand is unable to access the binding site of the target molecule, as shown in Figure 55a. In these circumstances a "spacer arm" is interposed between the matrix and the ligand to facilitate effective binding. Spacer arms must be designed to maximize binding, but to avoid non-specific binding effects. Figure 55 shows the improvement that can be seen in a purification as the spacer arm creates a more effective environment for binding. a) b) A 280 A 280 Efficient binding target elutes in a single peak 5 10 15 20 Inefficient binding target elutes during binding and elution 0 5 10 15 20 25 Elution volume, ml 0 25 Elution volume, ml Fig. 55. Using spacer arms. a) Ligand attached directly to the matrix. b) Ligand attached to the matrix via a spacer arm. The length of the spacer arm is critical. If it is too short, the arm is ineffective and the ligand fails to bind substances in the sample. If it is too long, proteins may bind nonspecifically to the spacer arm and reduce the selectivity of the separation. As a general rule, use spacer arms when coupling molecules M r < 1 000. Spacer arms are not generally needed for larger molecules. Table 7 shows the pre-activated media with different types of spacers arms that are available from Amersham Pharmacia Biotech. 98
Ligand coupling Several methods are available to couple a ligand to a pre-activated matrix. The correct choice of coupling method depends on the ligand characteristics. The use of commercially available, pre-activated media is recommended to save time and avoid the use of the potentially hazardous reagents that are required in some cases. Table 7. Examples of pre-activated media. NHS-activated Sepharose High Performance NHS-activated Sepharose 4 Fast Flow CNBr-activated Sepharose 4 Fast Flow EAH Sepharose 4B ECH Sepharose 4B Epoxy-activated Sepharose 6B Activated Thiol Sepharose 4B Thiopropyl Sepharose 6B 12-atom hydrophilic spacer arm to couple via amino groups. As above. Coupling via primary amino groups. 10-atom spacer arms to couple via amino groups. 9-atom spacer arms to couple via carboxyl groups. 12-atom hydrophilic spacer arm to couple through hydroxyl, amino or thiol groups. 10-atom spacer arm for reversible coupling through free thiol groups. 4-atom hydrophilic spacer arm for reversible coupling of proteins and small thiolated ligands through thiol groups. Also reacts with heavy metal ions, alkyl and aryl halides and undergoes addition reactions with compounds containing C=O, C=C and N=N bonds. Ligand specificity For purification of specific molecules or groups of molecules, many ligands are available coupled to an appropriate matrix (see Chapter 3). Ligands can also be isolated and purified to prepare a specific affinity medium for a specific target molecule. Coupling of ligands to pre-activated matrices is described in Chapter 5. 99
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Affinity Chromatography Principles
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Affinity Chromatography Principles
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Serine proteases and zymogens with
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Appendix 4 ........................
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This handbook describes the role of
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Affinity chromatography is also use
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BioProcess Media for large-scale pr
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Chapter 2 Affinity chromatography i
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Avoid using magnetic stirrers as th
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pH elution A change in pH alters th
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0.6 0.4 0.2 0 A280 pH selected for
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Situation Cause Remedy Protein does
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Chapter 3 Purification of specific
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Table 2. Relative binding strengths
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Purification examples Figure 11 sho
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MAbTrap Kit Fig. 13. MAbTrap Kit, r
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Media characteristics Ligand Compos
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A 280 nm 2.0 1.5 1.0 0.5 Column: rP
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Desalt and/or transfer purified IgG
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Performing a separation Column: Rec
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M r 97 000 66 000 45 000 30 000 20
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in The Recombinant Protein Handbook
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Cleaning These procedures are appli
Page 49 and 50: Purification examples Figures 24 an
Page 51 and 52: Nickel solution: 0.1 M NiSO 4 Bindi
Page 53 and 54: Protein A fusion proteins IgG Sepha
Page 55 and 56: Purification or removal of serine p
Page 57 and 58: Sample: 2 ml clarified E. coli homo
Page 59 and 60: Serine proteases and zymogens with
Page 61 and 62: DNA binding proteins HiTrap Heparin
Page 63 and 64: Sample: Column: Binding buffer: Elu
Page 65 and 66: Media characteristics Ligand densit
Page 67 and 68: Sample: Pooled and frozen human pla
Page 69 and 70: The harsh conditions required to br
Page 71 and 72: Purification or removal of albumin
Page 73 and 74: IFN-act. A units 280 nm 250 0.12 20
Page 75 and 76: Purification options Binding capaci
Page 77 and 78: Sepharose NH (CH 2) n NH N N O N N
Page 79 and 80: If detergent or denaturing agents h
Page 81 and 82: Glycoproteins or polysaccharides Co
Page 83 and 84: For complex samples containing glyc
Page 85 and 86: For complex samples containing glyc
Page 87 and 88: Chemical stability Avoid exposure t
Page 89 and 90: Proteins and peptides with exposed
Page 91 and 92: Several methods can be used to dete
Page 93 and 94: Media characteristics Composition M
Page 95 and 96: Performing a separation Binding buf
Page 97 and 98: Do not store the suspension for lon
Page 99: Sepharose has been modified and dev
Page 103 and 104: Couple a ligand through the least c
Page 105 and 106: Table 9 summarizes recommended liga
Page 107 and 108: Coupling through the primary amine
Page 109 and 110: Ligand and column preparation 1. Di
Page 111 and 112: Options Product Spacer Coupling con
Page 113 and 114: 100 80 Coupled substance, % 60 40 2
Page 115 and 116: Coupling small ligands through amin
Page 117 and 118: Ligand coupling 1. Add the ligand s
Page 119 and 120: Alternative coupling solutions: Dis
Page 121 and 122: Media characteristics Product Compo
Page 123 and 124: Ligands containing amino groups can
Page 125 and 126: Chapter 6 Affinity chromatography a
Page 127 and 128: Resolution is achieved by the selec
Page 129 and 130: For any capture step, select the te
Page 131 and 132: Appendix 1 Sample preparation Sampl
Page 133 and 134: Remove salts from proteins with mol
Page 135 and 136: 1. Filter (0.45 µm) or centrifuge
Page 137 and 138: For laboratory scale operations, Ta
Page 139 and 140: Removal of lipoproteins Lipoprotein
Page 141 and 142: Appendix 2 Selection of purificatio
Page 143 and 144: 4. Estimate the amount of slurry (r
Page 145 and 146: Appendix 5 Conversion data: protein
Page 147 and 148: Middle unit residue (-H 2 0) Charge
Page 149 and 150: Expected results when changing cond
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Expected results with competitive e
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Appendix 8 Analytical assays during
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Appendix 9 Storage of biological sa
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Ordering information Product Quanti
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STREAMLINE, Sepharose, HiTrap, ÄKT
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