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Purification options Binding capacity Maximum Comments operating flow HiTrap Benzamidine FF Trypsin, > 35 mg/column 4 ml/min (1 ml column) Prepacked columns**. (high sub) Trypsin, > 175 mg/column 15 ml/min (5 ml column) Benzamidine Sepharose 4 Trypsin, > 35 mg/ml medium 300 cm/h* Supplied as a Fast Flow (high sub) 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. **Supplied in 0.05 M acetate, pH 4 containing 20% ethanol. Purification examples Figure 30 shows an example of the removal of trypsin-like proteases from human plasma to prevent proteolysis of the plasma components, using a low pH elution. The activity test demonstrated that almost all trypsin-like protease activity is removed from the sample and bound to the column. Sample: 1 ml human plasma filtered through a 0.45 µm filter Column: HiTrap Benzamidine FF (high sub), 1 ml Binding buffer: 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4 Elution buffer: 50 mM glycine, pH 3.0 0–100% elution buffer in one step Flow: 1.0 ml/min System: ÄKTAexplorer Protease activity: S-2288 from Chromogenix, Heamochrom Diagnostica AB A 405 measurement. The activity is presented as the proteolytic activity/mg protein A 280 IU/litre A 405 3.0 1.00 A 280 2.5 0.80 2.0 0.60 1.5 0.40 1.0 0.20 0.5 0 0.0 5.0 10.0 15.0 ml Fig. 30. Removal of trypin-like serine proteases from human plasma using HiTrap Benzamidine FF (high sub), 1 ml. Figure 31 shows the effectiveness of using a GSTrap FF column with a HiTrap Benzamidine FF (high sub) for purification of a GST fusion protein, followed by cleavage of the GST tag via the thrombin cleavage site and subsequent removal of the thrombin enzyme. The GST fusion protein binds to the GSTrap FF column as other proteins wash through the column. Thrombin is applied to the column and incubated for 2 hours. A HiTrap Benzamidine FF (high sub) column, pre-equilibrated in binding buffer, is attached after the GSTrap FF column and both columns are washed in binding buffer followed by a high salt buffer. The cleaved protein and thrombin wash through from the GSTrap FF column, thrombin binds to the HiTrap Benzamidine FF (high sub) column, and the eluted fractions contain pure cleaved protein. 54
Sample: 2 ml clarified E. coli homogenate expressing a M r 37 000 SH2-GST fusion protein with a thrombin cleavage site Columns: GSTrap FF, 1 ml and HiTrap Benzamidine FF (high sub), 1 ml Binding buffer: 20 mM sodium phosphate, 0.15 M NaCl, pH 7.5 High salt wash buffer: 20 mM sodium phosphate, 1.0 M NaCl, pH 7.5 Benzamidine elution buffer: 20 mM p-aminobenzamidine in binding buffer GST elution buffer: 20 mM reduced glutathione, 50 mM Tris, pH 8.0 Flow: 0.5 ml/min System: ÄKTAprime Protease treatment: 20 units/ml thrombin (Amersham Pharmacia Biotech) for 2 hours at room temperature Thrombin activity: S-2238 (Chromogenix, Haemochrom Diagnostica AB) was used as a substrate and its absorbance at 405 nm was measured A 280 nm Thrombin High salt buffer wash Elution of HiTrap Benzamidine FF (high sub) Elution of GSTrap FF Thrombin activity A 405 nm 0.80 Thrombin GST-tag 0.30 0.60 0.40 0.20 0.20 fr.2 Cleaved SH2 protein fr.6 fr.7 fr.8 fr.14 fr.21 fr.22 0.10 0 0 0 10 15 20 25 50 ml A) B) A) B) A) B) A) GSTrap FF, 1 ml B) HiTrap Benzamidine FF (high sub), 1 ml M r 97 000 66 000 45 000 30 000 20 100 14 400 1 2 3 4 5 6 7 8 9 Gel: ExcelGel SDS Gradient 8–18%, Coomassie Blue staining Lane 1. Low Molecular Weight Calibration Kit (LMW) Lane 2. Clarified E. coli homogenate expressing SH2-GST fusion protein Lane 3. Flow-through from GSTrap FF (Fraction 2) Lane 4. SH2 GST-tag cleaved, washed off with binding buffer through both columns (Fraction 6) Lane 5. as above (Fraction 7) Lane 6. as above (Fraction 8) Lane 7. Elution of thrombin, HiTrap Benzamidine FF (high sub) Lane 8. Elution of GST-tag and some non-cleaved SH2-GST, GSTrap FF (Fraction 21) Lane 9. as above (Fraction 22) Fig. 31. On-column cleavage of a GST fusion protein and removal of thrombin after on-column cleavage, using GSTrap FF and HiTrap Benzamidine FF (high sub). Performing a separation Binding buffer: 0.05 M Tris-HCl, 0.5 M NaCl, pH 7.4 Elution buffer alternatives: - pH elution: 0.05 M glycine-HCl, pH 3.0 or 10 mM HCl, 0.05 M NaCl, pH 2.0 - competitive elution: 20 mM p-aminobenzamidine in binding buffer - denaturing eluents: 8 M urea or 6 M guanidine hydrochloride 55
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 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: Purification or removal of serine p
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
Page 107 and 108:
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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