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Sample: 2000 ml partially purified sample from DEAE Sepharose CL-4B flow-through, pH 7.0 Column: HiPrep 16/10 Heparin FF Binding buffer: 50 mM sodium phosphate, pH 7.5 Elution buffer: 50 mM sodium phosphate, 1 M sodium chloride, pH 7.5 Flow: 1.5 ml/min (45 cm/h) Chromatographic procedure: A ( ) Equilibration binding buffer: 80 ml Sample application: 2000 ml Wash with binding buffer: 100 ml Elution: 300 ml elution buffer as linear gradient 0–100% 1 1 fr. 13–14 280 fr. 23–24 scCro8, fr. 54–55 fr. 69 ( ) [NaCl] (M) M r 97 000 66 000 45 000 30 000 20 100 14 400 1 2 3 4 5 6 7 8 9 10 Electrophoresis: SDS-PAGE, 12% gel, Coomassie Blue staining Lane 1. Pool from HiPrep 26/10 Desalting Lane 2. Flow-through pool from DEAE Sepharose CL-4B Lane 3. Low Molecular Weight Calibration Kit (LMW) Lane 4-10. Eluted fractions from HiPrep 16/10 Heparin FF Lane 4. Fraction 13 Lane 5. Fraction 14 Lane 6. Fraction 23 Lane 7. Fraction 24 Lane 8. Fraction 54 Lane 9. Fraction 55 Lane 10. Fraction 69 0 0 Fig. 36. scCro8 purification on HiPrep 16/10 Heparin FF. Performing a separation Binding buffers: 20 mM Tris-HCl, pH 8.0 or 10 mM sodium phosphate, pH 7.0 Elution buffer: 20 mM Tris-HCl, 1–2 M NaCl, pH 8.0 or 10 mM sodium phosphate, 1–2 M NaCl, pH 7.0 1. Equilibrate the column with 10 column volumes of binding buffer. 2. Apply the sample. 3. Wash with 5–10 column volumes of binding buffer or until no material appears in the eluent (monitored by UV absorption at A 280 nm ). 4. Elute with 5–10 column volumes of elution buffer using a continuous or step gradient from 0–100% elution buffer. Modify the selectivity of heparin by altering pH or ionic strength of the buffers. Elute using a continuous or step gradient with NaCl, KCl or (NH 4 ) 2 SO 4 up to 1.5–2 M. Cleaning Remove ionically bound proteins by washing with 0.5 column volume 2 M NaCl for 10–15 minutes. Remove precipitated or denatured proteins by washing with 4 column volumes 0.1 M NaOH for 1–2 hours or 2 column volumes 6 M guanidine hydrochloride for 30–60 minutes or 2 column volumes 8 M urea for 30–60 minutes. Remove hydrophobically bound proteins by washing with 4 column volumes 0.1% – 0.5% Triton X-100 for 1–2 hours. 62
Media characteristics Ligand density Composition pH stability* Mean particle size HiTrap Heparin HP 10 mg/ml Heparin coupled to Short term 5–10 34 µm Sepharose High Performance Long term 5–10 using a N-hydroxysuccinimide coupling method to give stable attachment to the matrix through alkylamine and ether links. Heparin Sepharose 5 mg/ml Heparin coupled to Sepharose 6 Short term 4–13 90 µm 6 Fast Flow Fast Flow by reductive amination Long term 4–12 to give a stable attachment even HiPrep 16/10 in alkaline conditions. Heparin FF *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 0.1 M NaOH (1 week at +20 °C), 0.05 M sodium acetate, pH 4.0, 4 M NaCl, 8 M urea, 6 M guanidine hydrochloride. Storage Wash media and columns with 0.05 M sodium acetate containing 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C. 63
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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
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 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: Sample: Column: Binding buffer: Elu
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
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
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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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