Affinity Chromatography - Department of Molecular and Cellular ...

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Purification options Binding capacity/ml Maximum Comments medium operating flow Agarose Wheat Germ Lectin No data available 75 cm/h* Supplied as a suspension ready for column packing. *See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm. Performing a separation Binding buffer: 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4 Elution buffer: 0.5 M N-acetylglucosamine, 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4 Agarose Wheat Germ Lectin can be used with detergents, such as 1% deoxycholate or 0.5% Triton X-100. 1. Pack the column (see Appendix 3) and wash with at least 10 column volumes of binding buffer to remove preservative. 2. Equilibrate the column with 10 column volumes of binding buffer. 3. Apply the sample, using a low flow from 15 cm/h, during sample application (flow rate is the most significant factor for maximum binding). 4. Wash with 5–10 column volumes of binding buffer or until no material appears in the eluent (monitored by UV absorption at A 280 nm ). 5. Elute with 5 column volumes of elution buffer. Use 0–0.5 M N-acetylglucosamine, 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4 with a continuous gradient or step elution to improve resolution of complex samples containing glycoproteins with different affinities for the lectin. Elute tightly bound substances with 20 mM acetate buffer, pH 4.5 or with an alternative sugar, for example triacetylchitotriose. Higher concentrations of eluting substances may be necessary and recovery may be improved by pausing the flow for some minutes during elution. Cleaning Wash with 5–10 column volumes of 20 mM Tris-HCl, 1 M NaCl, pH 8.5 and re-equilibrate immediately with binding buffer. Low concentrations of non-ionic detergents in the Tris-HCl buffer can be used if necessary, for example 0.1% Nonidet P-40. Media characteristics Ligand density Composition pH stability* Mean particle size Agarose Wheat Germ Lectin 1–2 mg/ml Wheat Germ Lectin Short term 4–9 90 µm coupled to Sepharose 4B Long term 4–9 by CNBr method *Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. 84
Chemical stability Avoid exposure to conditions below pH 4.0 as this causes dissociation of the wheat germ lectin dimer. Storage Wash media and columns with 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C. Calmodulin binding proteins: ATPases, adenylate cyclases, protein kinases, phosphodiesterases, neurotransmitters Calmodulin Sepharose 4B Calmodulin is a highly conserved regulatory protein found in all eukaryotic cells. This protein is involved in many cellular processes such as glycogen metabolism, cytoskeletal control, neurotransmission, phosphate activity and control of NAD + /NADP + ratios. Calmodulin Sepharose 4B provides a convenient method for the isolation of many of the calmodulin binding proteins involved in these pathways. Calmodulin binds proteins principally through their interactions with hydrophobic sites on its surface. These sites are exposed after a conformational change induced by the action of Ca 2+ on separate Ca 2+ -binding sites. The binding of enzymes may be enhanced if the enzyme substrate is present and enzyme-substrate-calmodulin-Ca 2+ complexes are particularly stable. Purification options Binding capacity/ml Maximum Comments medium operating flow Calmodulin Sepharose 4B No data available 75 cm/h* Supplied as a suspension ready for column packing. *See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm. Performing a separation Binding buffer: 50 mM Tris-HCl, 0.05–0.2 M NaCl, 2 mM CaCl 2 , pH 7.5 Elution buffer: 50 mM Tris-HCl, 0.05–0.2 M NaCl, 2 mM EGTA, pH 7.5 1. Pack the column (see Appendix 3) and wash with at least 10 column volumes of binding buffer to remove preservative. 2. Equilibrate the column with 10 column volumes of binding buffer. 3. Apply the sample, using a low flow from 15 cm/h, during sample application (flow rate is the most significant factor for maximum binding). 4. Wash with 5–10 column volumes of binding buffer or until no material appears in the eluent (monitored by UV absorption at A 280 nm ). 5. Elute with 5 column volumes of elution buffer. 85
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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
Page 35 and 36: Media characteristics Ligand Compos
Page 37 and 38: A 280 nm 2.0 1.5 1.0 0.5 Column: rP
Page 39 and 40: Desalt and/or transfer purified IgG
Page 41 and 42: Performing a separation Column: Rec
Page 43 and 44: M r 97 000 66 000 45 000 30 000 20
Page 45 and 46: in The Recombinant Protein Handbook
Page 47 and 48: 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: For complex samples containing glyc
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 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
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For laboratory scale operations, Ta
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Removal of lipoproteins Lipoprotein
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Appendix 2 Selection of purificatio
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4. Estimate the amount of slurry (r
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Appendix 5 Conversion data: protein
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Middle unit residue (-H 2 0) Charge
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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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