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Table 3 gives examples of some typical binding and elution conditions that have been used with Protein A Sepharose. Table 3. Binding to free Protein A Sepharose Protein A Sepharose Species Subclass protein A binding pH elution pH Usually elutes by pH 3 Human IgG 1 + + 6.0–7.0 3.5–4.5 IgG 2 + + 6.0–7.0 3.5–4.5 IgG 3 – 8.0–9.0 < 7.0 IgG 4 + + 7.0–8.0 use step elution Cow IgG 2 + + 2 Goat IgG 2 + 5.8 Guinea pig IgG 1 + + 4.8 IgG 2 + + 4.3 Mouse IgG 1 + 8.0–9.0 5.5–7.5 IgG 2a + 7.0–8.0 4.5–5.5 IgG 2b + 7 3.5–4.5 IgG 3 + 7 4.0–7.0 Rat IgG 1 + > 9.0 7.0–8.0 IgG 2a – > 9.0 < 8.0 IgG 2b – > 9.0 < 8.0 IgG 3 + 8.0–9.0 3–4 (using thiocyanate) Binding strengths are tested with free protein A. They can be used as a guide to predict the binding behaviour to a protein A affinity medium. However, when coupled to an affinity matrix the interaction may be altered. For example, rat IgG 1 does not bind to protein A, but does bind to Protein A Sepharose. IgGs from most species and subclasses bind protein A near to physiological pH and ionic strength. Avoid excessive washing if the interaction between the protein of interest and the ligand is weak, since this may decrease the yield. With some antibodies, such as mouse IgG 1 , it might be necessary to add sodium chloride up to 3 M in the binding buffer to achieve efficient binding when using protein A, for example 1.5 M glycine, 3 M NaCl, pH 8.9. Alternative elution buffers include: 1 M acetic acid, pH 3.0 or 0.1 M glycine-HCl, pH 3.0 or 3 M potassium isothiocyanate. Potassium isothiocyanate can severely affect structure and immunological activity. Use a mild elution method when labile antibodies are isolated. Reverse the flow of the wash buffer and elute with 0.1 M glycyltyrosine in 2 M NaCl, pH 7.0 at room temperature, applied in pulses. (Note: glycyltyrosine absorbs strongly at wavelengths used for detecting proteins). The specific elution is so mild that the purified IgG is unlikely to be denatured. To increase capacity, connect several HiTrap Protein A HP or HiTrap rProtein A FF columns (1 ml or 5 ml) in series or pack a larger column with Protein A Sepharose 4 Fast Flow or rProtein A Sepharose 4 Fast Flow (see Appendix 3). 36
Desalt and/or transfer purified IgG fractions into a suitable buffer using a desalting column (see page 134). Reuse of Protein A Sepharose and rProtein A Sepharose media depends on the nature of the sample and should only be performed with identical samples to prevent cross-contamination. Media characteristics Product Ligand Composition pH stability* Mean particle density size HiTrap Protein A HP 3 mg/ml Ligand coupled to Short term 2–10 34 µm Sepharose HP by Long term 3–9 N-hydroxysuccinimide activation (stable attachment through alkylamine and ether links). Protein A Sepharose 4 6 mg/m Ligand coupled to Short term 2–10 90 µm Fast Flow** Sepharose 4 Fast Flow Long term 3–9 by cyanogen bromide activation. HiTrap rProtein A FF 6 mg/ml Ligand coupled to Short term 2–11 90 µm Sepharose 4 Fast Flow Long term 3–10 by epoxy activation, thioether coupling. rProtein A Sepharose 4 6 mg/ml Ligand coupled to Sepharose 4 Short term 2–11 90 µm Fast Flow** Fast Flow by epoxy activation, Long term 3–10 thioether coupling. *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. **Protein A Sepharose 4 Fast Flow and rProtein A Sepharose 4 Fast Flow have a higher binding capacity, a more rigid matrix and provide more convenient alternatives to Protein A Sepharose CL-4B which must be rehydrated before column packing. Chemical stability These media and columns tolerate high concentrations of urea, guanidine HCl and chaotropic agents. Storage Wash media and columns with 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C. 37
Page 1 and 2: Affinity Chromatography Principles
Page 3 and 4: Affinity Chromatography Principles
Page 5 and 6: Serine proteases and zymogens with
Page 7: Appendix 4 ........................
Page 10 and 11: This handbook describes the role of
Page 12 and 13: Affinity chromatography is also use
Page 14 and 15: BioProcess Media for large-scale pr
Page 17 and 18: Chapter 2 Affinity chromatography i
Page 19 and 20: Avoid using magnetic stirrers as th
Page 21 and 22: pH elution A change in pH alters th
Page 23 and 24: 0.6 0.4 0.2 0 A280 pH selected for
Page 25 and 26: Situation Cause Remedy Protein does
Page 27 and 28: Chapter 3 Purification of specific
Page 29 and 30: Table 2. Relative binding strengths
Page 31 and 32: Purification examples Figure 11 sho
Page 33 and 34: MAbTrap Kit Fig. 13. MAbTrap Kit, r
Page 35 and 36: Media characteristics Ligand Compos
Page 37: A 280 nm 2.0 1.5 1.0 0.5 Column: rP
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 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 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
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Coupling through the primary amine
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Ligand and column preparation 1. Di
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Options Product Spacer Coupling con
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100 80 Coupled substance, % 60 40 2
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Coupling small ligands through amin
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Ligand coupling 1. Add the ligand s
Page 119 and 120:
Alternative coupling solutions: Dis
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Media characteristics Product Compo
Page 123 and 124:
Ligands containing amino groups can
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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
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Remove salts from proteins with mol
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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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