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Media characteristics Ligand and density Composition pH stability* Mean particl size HiTrap Blue HP Cibacron Blue F3G-A Ligand coupled to Short term 3–13 34 µm 4 mg/ml Sepharose High Long term 4–12 Performance using the triazine method. Blue Sepharose 6 Cibacron Blue F3G-A Ligand coupled to Short term 3–13 90 µm Fast Flow 6.7–7.9 µmoles/ml Sepharose Fast Flow Long term 4–12 using the triazine 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. Chemical stability Stable in all commonly used aqueous buffers, 70% ethanol, 8 M urea and 6 M guanidine hydrochloride. Storage Wash media and columns with 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C. NAD+-dependent dehydrogenases and ATP-dependent kinases 5' AMP Sepharose 4B, HiTrap Blue HP, Blue Sepharose 6 Fast Flow NAD + -dependent dehydrogenases and ATP-dependent kinases interact strongly with 5' AMP so that selective elution with gradients of NAD + or NADP + enables the resolution of complex mixtures of dehydrogenase isoenzymes, using 5' AMP Sepharose 4B. Synthesis of 5' AMP Sepharose 4B takes place in several steps. Diaminohexane is linked to AMP via the N 6 of the purine ring. The derivatized AMP is then coupled to Sepharose 4B via the aminohexane spacer. NAD + -dependent dehydrogenases and ATP-dependent kinases are also members of a larger group of proteins that will interact with Cibacron Blue F3G-A, a synthetic polycyclic dye that shows certain structural similarities to the cofactor NAD + . When used as an affinity ligand attached to Sepharose 6 Fast Flow or Sepharose HP, Cibacron Blue F3G-A will bind strongly and specifically to a wide range of proteins. Some proteins bind specifically due to their requirement for nucleotide cofactors, while others, such as albumin, lipoproteins, blood coagulation factors and interferon, bind in a less specific manner by electrostatic and/or hydrophobic interactions with the aromatic anionic ligand. 72
Purification options Binding capacity Maximum Comments operating flow 5' AMP Sepharose 4B Lactate dehydrogenase, 75 cm/h* High specificity for 10 mg/ml medium proteins with affinity (0.1 M phosphate buffer, for NAD + . Supplied as pH 7.0 at +20 °C) a freeze-dried powder, rehydration required. HiTrap Blue HP Human serum albumin, 4 ml/min (1 ml column) General specificity for 20 mg/column proteins with affinity for Human serum albumin, 20 ml/min (5 ml column) NADP + and other proteins 100 mg/column that react less specifically. Prepacked columns. Blue Sepharose 6 Human serum albumin, 400 cm/h* General specificity for Fast Flow > 18 mg/ml medium proteins with affinity for NADP + and other proteins that react less specifically. 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. 5' AMP Sepharose 4B Performing a separation Swell the required amount of powder for 15 min. in 0.1 M phosphate buffer, pH 7.0 (100 ml per gram dry powder) and wash on a sintered glass filter. Pack the column (see Appendix 3). Binding buffer: 10 mM phosphate, 0.15 M NaCl, pH 7.3 If the protein of interest binds to the medium via ionic forces, it may be necessary to reduce the concentration of NaCl in the binding buffer. Elution buffers: • use low concentrations of the free cofactor, NAD + or NADP + (up to 20 mM) with step or gradient elution. If detergent or denaturing agents have been used during purification, these can also be used in the high and low pH wash buffers. Cleaning Wash 3 times with 2–3 column volumes of buffers, alternating between high pH (0.5 M NaCl, 0.1 M Tris-HCl, pH 8.5) and low pH (0.5 M NaCl, 0.1 M sodium acetate, pH 4.5). Re-equilibrate immediately with 3–5 column volumes of binding buffer. Remove denatured proteins or lipids by washing the column with 2 column volumes of detergent e.g. 0.1% Triton X-100 for 1 minute. Re-equilibrate immediately with 5 column volumes of binding buffer. 73
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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
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 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: IFN-act. A units 280 nm 250 0.12 20
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
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
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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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