Affinity Chromatography - Department of Molecular and Cellular ...

More documents

Recommendations

Info

Remove proteases as quickly as possible from the sample as the calmodulin-binding sites on proteins are frequently very susceptible to protease action (see page 53). Remove free calmodulin from the sample by hydrophobic interaction chromatography in the presence of Ca 2+ on HiTrap Phenyl FF (high sub) or by ion exchange chromatography on HiTrap Q FF. Since some non-specific ionic interactions can occur, a low salt concentration (0.05–0.20 M NaCl) is recommended to promote binding to the ligand while eliminating any non-specific binding. Use chelating agents to elute the proteins. Chelating agents strip Ca 2+ from the calmodulin, reversing the conformational change that exposed the protein binding sites. Calcium ions may also be displaced by a high salt concentration, 1 M NaCl. Cleaning Alternative 1 Wash with 3 column volumes of 0.05 M Tris-HCl, 1.0 M NaCl, 2 mM EGTA, pH 7.5 and re-equilibrate immediately with 5–10 column volumes of binding buffer. Alternative 2 Wash with 3 column volumes of 0.1 M ammonium carbonate buffer, 2 mM EGTA, pH 8.6 followed by 3 column volumes of 1 M NaCl, 2 mM CaCl 2 . Continue washing with 3 column volumes of 0.1 M sodium acetate buffer, 2 mM CaCl 2 , pH 4.4 followed by 3 column volumes of binding buffer. Remove severe contamination by washing with non-ionic detergent such as 0.1% Triton X-100 at +37 °C for 1 min. Media characteristics Ligand density Composition pH stability* Mean particle size Calmodulin Sepharose 4B 0.9–1.3 mg/ml Bovine testicular calmodulin Short term 4–9 90 µm coupled to Sepharose 4B by Long term 4–9 the CNBr 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 solutions. Storage Wash media and columns with 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C. 86
Proteins and peptides with exposed amino acids: His, Cys, Trp, and/or with affinity for metal ions (also known as IMAC, immobilized metal chelate affinity chromatography) HiTrap Chelating HP, Chelating Sepharose Fast Flow, His MicroSpin Purification Module, HisTrap Kit Proteins and peptides that have an affinity for metal ions can be separated using metal chelate affinity chromatography. The metals are immobilized onto a chromatographic medium by chelation. Certain amino acids, e.g. histidine and cysteine, form complexes with the chelated metals around neutral pH (pH 6–8) and it is primarily the histidine-content of a protein which is responsible for its binding to a chelated metal. Metal chelate affinity chromatography is excellent for purifying recombinant (His) 6 fusion proteins (see page 46) as well as many natural proteins. Chelating Sepharose, the medium used for metal chelate affinity chromatography, is formed by coupling a metal chelate forming ligand (iminodiacetic acid) to Sepharose. Before use the medium is loaded with a solution of divalent metal ions such as Ni 2+ , Zn 2+ , Cu 2+ , Ca 2+ , Co 2+ or Fe 2+ . The binding reaction with the target protein is pH dependent and bound sample is, most commonly, eluted by reducing the pH and increasing the ionic strength of the buffer or by including EDTA or imidazole in the buffer. The structure of the ligand, iminodiacetic acid, is shown in Figure 47. S epharose CH 2 COOH O CH2 CH CH2 O CH2 CH CH2 N OH OH CH 2 COOH Fig. 47. Partial structure of Chelating Sepharose High Performance and Chelating Sepharose Fast Flow. Metalloproteins are not usually suitable candidates for purification by chelating chromatography since they tend to scavenge the metal ions from the column. Purification options Binding capacity Maximum Comments operating flow His MicroSpin Purification 100 µg/column Not applicable Ready to use, prepacked columns, Module buffers and chemicals for purification of (His) 6 fusion proteins. HiTrap Chelating HP 1 ml 12 mg/column 4 ml/min Prepacked column, ready to use. HiTrap Chelating HP 5 ml 60 mg/column 20 ml/min Prepacked column, ready to use. HisTrap Kit 12 mg/column* 4 ml/min Ready to use, prepacked columns, buffers and chemicals for purification of (His) 6 fusion proteins for up to 12 purifications using a syringe. Chelating Sepharose 12 mg/ml medium 400 cm/h** Supplied as suspension for packing Fast Flow columns and scale up. *Estimate for a (His) 6 fusion protein of M r 27 600, binding capacity varies according to specific protein. **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. 87
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 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
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: Chemical stability Avoid exposure t
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
Page 125 and 126: Chapter 6 Affinity chromatography a
Page 127 and 128: Resolution is achieved by the selec
Page 129 and 130: For any capture step, select the te
Page 131 and 132: Appendix 1 Sample preparation Sampl
Page 133 and 134: Remove salts from proteins with mol
Page 135 and 136: 1. Filter (0.45 µm) or centrifuge
Page 137 and 138: For laboratory scale operations, Ta
Page 139 and 140:
Removal of lipoproteins Lipoprotein
Page 141 and 142:
Appendix 2 Selection of purificatio
Page 143 and 144:
4. Estimate the amount of slurry (r
Page 145 and 146:
Appendix 5 Conversion data: protein
Page 147 and 148:
Middle unit residue (-H 2 0) Charge
Page 149 and 150:
Expected results when changing cond
Page 151 and 152:
Expected results with competitive e
Page 153 and 154:
Appendix 8 Analytical assays during
Page 155 and 156:
Appendix 9 Storage of biological sa
Page 157 and 158:
Ordering information Product Quanti
Page 159 and 160:
STREAMLINE, Sepharose, HiTrap, ÄKT
show all

Affinity Chromatography - Department of Molecular and Cellular ...

Create successful ePaper yourself

Delete template?

Save as template?