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Unexpected results - slow binding (adsorption) A 280 Flow through (unbound material) Elution buffer Some of the target elutes under binding conditions as a broad, low peak. Binding buffer Eluted target - Apply sample in aliquots to allow time for binding to take place. Slow on Fast off ml Unexpected results - slow elution (desorption) A 280 Binding buffer Flow through (unbound material) Elution buffer Eluted target Target elutes as a long, low peak. - Change elution scheme. - Use pulsed elution (see page 22). Fast on Slow off ml 150
Appendix 8 Analytical assays during purification Analytical assays are essential to follow the progress of purification. They are used to assess the effectiveness of each step in terms of yield, biological activity, recovery and to help during optimization of experimental conditions. The importance of a reliable assay for the target molecule cannot be over-emphasized. When testing chromatographic fractions, ensure that the buffers used for purification do not interfere with the assay. Total protein determination Lowry or Bradford assays are used most frequently to determine the total protein content. The Bradford assay is particularly suited to samples where there is a high lipid content that may interfere with the Lowry assay. Purity determination Purity is most often estimated by SDS-PAGE. Alternatively, isoelectric focusing, capillary electrophoresis, reversed phase chromatography or mass spectrometry may be used. SDS-PAGE Analysis Reagents Required 6X SDS loading buffer: 0.35 M Tris-HCl (pH 6.8), 10.28% (w/v) SDS, 36% (v/v) glycerol, 0.6 M dithiothreitol (or 5% 2-mercaptoethanol), 0.012% (w/v) bromophenol blue. Store in 0.5 ml aliquots at -80 °C. 1. Add 2 µl of 6X SDS loading buffer to 5–10 µl of supernatant from crude extracts, cell lysates or purified fractions as appropriate. 2. Vortex briefly and heat for 5 minutes at +90 to +100 °C. 3. Load the samples onto an SDS-polyacrylamide gel. 4. Run the gel and stain with Coomassie Blue (Coomassie Blue R Tablets) or silver (PlusOne Silver Staining Kit, Protein). The percentage of acrylamide in the SDS-gel should be selected according to the expected molecular weight of the protein of interest (see Table 19). Table 19. % Acrylamide in resolving gel Separation size range Single percentage: 5% 36 000–200 000 7.5% 24 000–200 000 10% 14 000–200 000 12.5% 14 000–100 000 15% 14 000–60 000 1 Gradient: 5–15% 14 000–200 000 5–20% 10 000–200 000 10–20% 10 000–150 000 1 The larger proteins fail to move significantly into the gel. 151
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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
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Several methods can be used to dete
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Media characteristics Composition M
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Performing a separation Binding buf
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Do not store the suspension for lon
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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 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
Page 151: Expected results with competitive e
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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