Affinity Chromatography Handbook

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48 Purification examples Figures 25 and 26 show the purification of recombinant proteins expressed in soluble form or as inclusion bodies and Figure 27 gives an example of simultaneous on-column purification and refolding of a recombinant protein expressed as an inclusion body. Sample: 9 ml E. coli periplasm containing Protein A-(HisGly) 4His. Diluted with 9 ml binding buffer. Column: HiTrap Chelating HP, 5 ml Metal ion: Zn 2+ Soluble recombinant proteins Flow: 1.0 ml/min Binding buffer: 50 mM sodium phosphate, 0.1 M NaCl, pH 8.0 Elution buffer: 50 mM sodium phosphate, 0.1 M NaCl, pH 4.0 Gradient: 20 ml elution buffer, step gradient Electrophoresis: SDS-PAGE, PhastSystem, PhastGel Gradient 8–25, 1 µl sample, Coomassie stained M r 97 000 66 000 45 000 30 000 20 100 14 400 1 2 3 Sample: 8 ml cell extract containing (His) 10-tagged protein. The clone was a kind gift from Dr. C. Fuller and S. Brasher, Department of Biochemistry, University of Cambridge, UK. Column: HiTrap Chelating HP, 1 ml Metal ion: Ni 2+ Recombinant protein expressed in inclusion bodies Binding buffers: 20 mM sodium phosphate, 0.5 M NaCl, 100 mM imidazole, 8 M urea or 6 M guanidine hydrochloride, pH 7.4 Elution buffers: 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, 8 M urea or 6 M guanidine hydrochloride, pH 7.4 Flow: Approx. 4 ml/min Equipment: Syringe Electrophoresis: SDS-PAGE, PhastSystem, PhastGel 10–15, 1 µl sample, silver staining Lane 1. Low Molecular Weight Calibration Kit (LMW), reduced Lane 2. Crude periplasmic fraction, reduced Lane 3. Pool I, purified Protein A-(HisGly) 4His, reduced Purification in 8 M urea M r 97 000 66 000 45 000 30 000 20 100 14 400 Purification in 6 M guanidine hydrochloride M r 97 000 66 000 45 000 30 000 20 100 14 400 A280 nm 0.10 Fig. 25. Purification of recombinant proteins on HiTrap Chelating HP, 5 ml, charged with Zn 2+ . 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Lane 1. Low Molecular Weight Calibration Kit (LMW) Lane 2. Starting material, cell extract, diluted 20-fold Lane 3. Flow-through, diluted 10-fold Lane 4. Wash Lane 5. Elution (first two ml) Lane 6. Elution (last two ml) Lane 7. LMW Lane 1. Low Molecular Weight Calibration Kit (LMW) Lane 2. Starting material, cell extract, diluted 10-fold Lane 3. Flow-through Lane 4. Wash Lane 5. Elution (first two ml) Lane 6. Elution (last two ml) Lane 7. LMW Fig. 26. Purification of (His) 10 -tagged protein from inclusion bodies on HiTrap Chelating HP, 1 ml, charged with Ni 2+ . 0.05 0 pool I 45 65 ml
One step, on-column, refolding and purification of recombinant proteins from inclusion bodies Column: Ni 2+ -loaded HiTrap Chelating HP, 1 ml Sample: N-terminal (His) 6 recombinant protein produced in E. coli Flow: 0.1–1 ml/min, sample loading and refolding 1 ml/min, wash and elution Binding buffer: 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 6 M guanidine hydrochloride, 1 mM 2-mercaptoethanol, pH 8.0 Washing buffer: 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 6 M urea, 1 mM 2-mercaptoethanol, pH 8.0 Refolding buffer: 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0 Refolding gradient: 30 ml Elution buffer: 20 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0 Elution gradient: 10 ml Fraction volumes: 3 ml sample loading, wash and refolding, 1 ml elution M r 97 000 66 000 45 000 30 000 20 100 14 400 1 2 3 4 5 6 7 8 M r 97 000 66 000 45 000 30 000 20 100 14 400 1 2 3 4 5 6 7 8 Lane 1. LMW Lane 2. Fraction 38 Lane 3. Fraction 39 Lane 4. Fraction 40 Lane 5. Fraction 41 Lane 6. Fraction 42 Lane 7. Fraction 46 Lane 8. Fraction 49 Fig. 27. One step refolding and purification of a (His) 6 -tagged recombinant protein on HiTrap Chelating HP, 1 ml, charged with Ni 2+ . The sample is bound to the column and all unbound material is washed through. Refolding of the bound protein is performed by running a linear 6–0 M urea gradient, starting with the wash buffer and finishing with the refolding buffer. A gradient volume of 30 ml or higher and a flow rate of 0.1–1 ml/min can be used. The optimal refolding rate should be determined experimentally for each protein. The refolded recombinant protein is eluted using a 10–20 ml linear gradient starting with refolding buffer and ending with the elution buffer. Performing a separation Figure 28 shows the simplicity of a poly (His) fusion protein purification when using a prepacked HiTrap Chelating HP column. The protocol described has been optimized for a high yield purification of (His) 6 fusion proteins and can be used as a base from which to scale up. An alternative optimization protocol designed to achieve high purity is supplied with the HisTrap Kit and is also described in The Recombinant Protein <strong>Handbook</strong>: Protein Amplification and Simple Purification from Amersham Biosciences. Prepare column Wash with H 2O Load with NiSO4 Wash with H 2O 3 min Lane 1. Low Molecular Weight Calibration Kit (LMW) Lane 2. Starting material for HiTrap Chelating HP, 1 ml Lane 3. Fraction 1 Gua-HCl wash (manually) Lane 4. Fraction 2 Gua-HCl wash (manually) Lane 5. Fraction 3 Gua-HCl wash (manually) Lane 6. Fraction 4 Gua-HCl wash (manually) Lane 7. Fraction 1 Urea wash (manually) Lane 8. Fraction 2 Urea wash (manually) Equilibrate column with binding buffer 3 min 5-15 min A 280 1.0 0.75 0.5 0.25 0 Start refolding Manually using a syringe: • Sample loading Gua-HCl wash Urea wash Apply sample Wash with binding buffer Start elution fr. fr. fr. 38 40 42 fr. 46 fr. 49 10 20 30 40 50 60 65 ml Electrophoresis: SDS-PAGE. PhastSystem, PhastGel 10–15, reducing conditions, 1 µl sample, Coomassie Blue staining. 2 min Elute with elution buffer Waste Waste Collect Collect fractions Fig. 28. HiTrap Chelating HP and a schematic overview of poly (His) fusion protein purification. 49
Page 1 and 2: 18-1022-29 Edition AD Affinity Chro
Page 3 and 4: Affinity Chromatography Principles
Page 5 and 6: DNA binding proteins ..............
Page 7: Appendix 4 ........................
Page 10 and 11: 8 This handbook describes the role
Page 12 and 13: 10 Affinity chromatography is also
Page 14 and 15: 12 BioProcess Media for large-scale
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: A 280 nm 0.6 0.4 0.2 0 Fig. 8. Scou
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 0.0 0 200
Page 39 and 40: Desalt and/or transfer purified IgG
Page 41 and 42: Performing a separation Column: HiT
Page 43 and 44: Fig. 21. SDS-PAGE of non-reduced sa
Page 45 and 46: Purification options Binding capaci
Page 47 and 48: Performing a separation Binding buf
Page 49: Poly (His) fusion proteins His Micr
Page 53 and 54: Purification using HisTrap Kit HisT
Page 55 and 56: 1. Pack the column (see Appendix 3)
Page 59 and 60: 1. Equilibrate the column with 5 co
Page 61 and 62: Specifically bound biomolecules can
Page 65 and 66: Sample: 2000 ml partially purified
Page 67 and 68: Coagulation factors HiTrap Heparin
Page 71 and 72: Purification or removal of fibronec
Page 73 and 74: Purification examples Figure 42 sho
Page 75 and 76: Media characteristics Ligand and de
Page 77 and 78: Media characteristics Ligand densit
Page 81 and 82: Elution buffers: use low concentrat
Page 85 and 86: Lentil lectin for binding of branch
Page 89 and 90: Remove proteases as quickly as poss
Page 91 and 92: Purification example A 280 nm 1.0 0
Page 93 and 94: Samples: 2.5 ml cell extract contai
Page 95 and 96: In Activated Thiol-Sepharose 4B the
Page 97 and 98: Reactivation Pass one to two column
Page 99 and 100: Chapter 4 Components of an affinity
Page 101 and 102:
If several functional groups are av
Page 103 and 104:
Chapter 5 Designing affinity media
Page 105 and 106:
Coupling the ligand 1. Prepare the
Page 107 and 108:
Increase the ligand concentration t
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Options Product Spacer arm Coupling
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Media characteristics Product Ligan
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Preparation of the matrix should be
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Immunoaffinity chromatography Immun
Page 117 and 118:
Preparation of coupling reagent Use
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Coupling through hydroxy, amino or
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Coupled glycylleucine % Coupled lig
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Coupling through a thiol group Thio
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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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Sample clarification Centrifugation
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Examples of precipitation agents ar
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Resolubilization of protein precipi
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Alternative 1: Manual desalting wit
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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 20) 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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Additional reading Code No. Purific
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Product Quantity Code No. Blue Seph
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STREAMLINE, Sepharose, HiTrap, ÄKT
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