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Antibody classes Characteristic Heavy chain Light chain IgG IgM g m a e d k or l k or l k or l k or l k or IgA IgE IgD l Y structure Fig. 10. Antibody classes. IgG, IgG fragments and subclasses The basis for purification of IgG, IgG fragments and subclasses is the high affinity of protein A and protein G for the Fc region of polyclonal and monoclonal IgG-type antibodies, see Figure 9. Protein A and protein G are bacterial proteins (from Staphylococcus aureus and Streptococcus, respectively) which, when coupled to Sepharose, create extremely useful, easy to use media for many routine applications. Examples include the purification of monoclonal IgG-type antibodies, purification of polyclonal IgG subclasses, and the adsorption and purification of immune complexes involving IgG. IgG subclasses can be isolated from ascites fluid, cell culture supernatants and serum. Table 2 shows a comparison of the relative binding strengths of protein A and protein G to different immunoglobulins compiled from various publications. A useful reference on this subject is also: Structure of the IgG-binding regions of streptococcal Protein G, EMBO J., 5, 1567–1575 (1986). Binding strengths are tested with free protein A or protein G and can be used as a guide to predict the binding behaviour to a protein A or protein G purification 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. 26
Table 2. Relative binding strengths of protein A and protein G to various immunoglobulins. No binding: -, relative strength of binding: +, ++, +++, ++++. Protein A Protein G Species Subclass binding binding Human IgA variable - IgD - - IgE IgG 1 ++++ ++++ IgG 2 ++++ ++++ IgG 3 - ++++ IgG 4 ++++ ++++ IgM A variable - Chicken IgY - - Avian egg yolk IgY B - - Cow ++ ++++ Dog ++ + Goat - ++ Guinea pig IgG 1 ++++ ++ IgG 2 ++++ ++ Hamster + ++ Horse ++ ++++ Koala - + Llama - + Monkey (rhesus) ++++ ++++ Mouse IgG 1 + ++++ IgG 2a ++++ ++++ IgG 2b +++ +++ IgG 3 ++ +++ IgM A variable - Pig +++ +++ Rabbit no distinction ++++ +++ Rat IgG 1 - + IgG 2a - ++++ IgG 2b - ++ IgG 3 + ++ Sheep +/- ++ A Purify using HiTrap IgM Purification HP columns. B Purify using HiTrap IgY Purification HP columns. Single step purification based on Fc region specificity will co-purify host IgG and may even bind trace amounts of serum proteins. For any preparation that must be free of even trace amounts of contaminating IgG, immunospecific affinity using anti-host IgG antibodies as the ligand to remove host IgG or using target specific antigen to avoid binding host IgG, ion exchange and/or hydrophobic interaction chromatography may be better alternatives (see Chapter 6). Both protein A and a recombinant protein A are available, with similar specificities for the Fc region of IgG. The recombinant protein A has been engineered to include a C-terminal cysteine that enables a single-point coupling to Sepharose. Single point coupling often results in an enhanced binding capacity. Genetically engineered antibodies and antibody fragments can have altered biological properties and also altered properties to facilitate their purification. For example, tags can be introduced into target molecules for which no affinity media were previously available thus creating a fusion protein that can be effectively purified by affinity chromatography. Details for the purification of tagged proteins are covered in the section Recombinant Fusion Proteins on page 42 of this handbook. For information on the purification of 27
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: Chapter 3 Purification of specific
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 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
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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
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Ligand coupling Several methods are
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Couple a ligand through the least c
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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
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Alternative coupling solutions: Dis
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Media characteristics Product Compo
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Ligands containing amino groups can
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Chapter 6 Affinity chromatography a
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Resolution is achieved by the selec
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For any capture step, select the te
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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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