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Appendix 3 Column packing and preparation Prepacked columns from Amersham Pharmacia Biotech will ensure reproducible results and the highest performance. However, if column packing is required, the following guidelines will apply at any scale of operation: With a high binding capacity medium, short, wide columns can be used for rapid purification, even with low linear flow rates. Ready to use affinity media are supplied with details of the binding capacity per ml of medium. Unless otherwise stated, estimate the amount of medium required to bind the target molecules and use two- to five times this amount to pack the column. Refer to the product instructions for more specific information regarding buffers, flow rates etc. For affinity media made from pre-activated matrices, determine the binding capacity of the medium. Estimate the amount of medium required to bind the target molecules and use two- to five times this amount to pack the column. A Column Packing Video is available to demonstrate how to produce a well-packed column (see Ordering information). The video focuses particularly on the importance of column packing for gel filtration, shown in the figure below. 1. Equilibrate all materials to the temperature at which the purification will be performed. 2. Eliminate air by flushing column end pieces with the recommended buffer. Ensure no air is trapped under the column net. Close column outlet leaving 1–2 cm of buffer in the column. 3. Gently resuspend the purification medium. For media not supplied in suspension, use a medium: buffer ratio of approximately 1:2 to produce a suspension for mixing during rehydration. Avoid using magnetic stirrers since they may damage the matrix. 140
4. Estimate the amount of slurry (resuspended medium) required on the basis of the recommendations supplied. 5. Pour the required volume of slurry into the column. Pouring down a glass rod held against the wall of the column will minimize the introduction of air bubbles. 6. Immediately fill the column with buffer. 7. Mount the column top piece and connect to a pump. 8. Open the column outlet and set the pump to the desired flow rate. If the recommended flow rate cannot be obtained, use the maximum flow rate the pump can deliver. Do not exceed the maximum operating pressure of the medium or column. 9. Maintain the packing flow rate for at least 3 column volumes after a constant bed height is obtained. Mark the bed height on the column. Do not exceed 75% of the packing flow rate during any purification. 10. Stop the pump and close the column outlet. Remove the top piece and carefully fill the rest of the column with buffer to form an upward meniscus at the top. 11. Insert the adaptor into the column at an angle, ensuring that no air is trapped under the net. 12. Slide the adaptor slowly down the column (the outlet of the adaptor should be open) until the mark is reached. Lock the adaptor in position. 13. Connect the column to the pump and begin equilibration. Re-position the adaptor if necessary. The medium must be thoroughly washed to remove the storage solution, usually 20% ethanol. Residual ethanol may interfere with subsequent procedures. Many media equilibrated with sterile PBS containing an antimicrobial agent may be stored at +4 °C for up to 1 month, but always follow the specific storage instructions supplied with the product. Columns for packing affinity media XK columns are fully compatible with the high flow rates achievable with modern media and a broad range of column dimensions is available. Columns XK 16/20 and XK 26/20 are recommended for affinity chromatography and their specifications are shown in Table 18. For a complete listing refer to the Amersham Pharmacia Biotech BioDirectory or web catalogue (www.apbiotech.com). In most cases, the column size required is governed by the capacity of the medium and the amount of substance to be purified. Table 18. Columns Volume (ml) Code no XK 16/20 2–34 18-8773-01 XK 26/20 0–80 18-1000-72 XK 50/20 0–275 18-1000-71 141
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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
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Purification examples Figures 24 an
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Nickel solution: 0.1 M NiSO 4 Bindi
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Protein A fusion proteins IgG Sepha
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Purification or removal of serine p
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Sample: 2 ml clarified E. coli homo
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Serine proteases and zymogens with
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DNA binding proteins HiTrap Heparin
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Sample: Column: Binding buffer: Elu
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Media characteristics Ligand densit
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Sample: Pooled and frozen human pla
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The harsh conditions required to br
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Purification or removal of albumin
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IFN-act. A units 280 nm 250 0.12 20
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Purification options Binding capaci
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Sepharose NH (CH 2) n NH N N O N N
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If detergent or denaturing agents h
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Glycoproteins or polysaccharides Co
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For complex samples containing glyc
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For complex samples containing glyc
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Chemical stability Avoid exposure t
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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 and 100: Sepharose has been modified and dev
Page 101 and 102: Ligand coupling Several methods are
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: Appendix 2 Selection of purificatio
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
Page 151 and 152: Expected results with competitive e
Page 153 and 154: Appendix 8 Analytical assays during
Page 155 and 156: Appendix 9 Storage of biological sa
Page 157 and 158: Ordering information Product Quanti
Page 159 and 160: STREAMLINE, Sepharose, HiTrap, ÄKT
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