Affinity Chromatography - Department of Molecular and Cellular ...

More documents

Recommendations

Info

Media characteristics Product Ligand density Composition pH stability* Mean particle size HiTrap NHS-activated HP 10 µmoles/ml 6-aminohexanoic acid Short term 3–12 34 µm linked by epoxy coupling to Long term 3–12 highly cross linked agarose, terminal carboxyl group esterified with NHS. NHS-activated 16–23 As above Short term 3–13 90 µm Sepharose 4 Fast Flow µmoles/ml Long term 3–13 *Long term refers to the pH interval over which the matrix 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. Stability data refers to the coupled medium provided that the ligand can withstand the pH. Storage Store the column in a solution that maintains the stability of the ligand and contains a bacteriostatic agent, for example PBS, 0.05% NaN 3 , pH 7.2. pH stability of the media when coupled to the chosen ligand will depend upon the stability of the ligand itself. Sodium azide can interfere with many coupling methods and some biological assays. It can be removed by using a desalting column (see page 134). CNBr-activated Sepharose CNBr-activated Sepharose offers a well-established option for the attachment of larger ligands and as an alternative to NHS-activated Sepharose. Cyanogen bromide reacts with hydroxyl groups on Sepharose to form reactive cyanate ester groups. Proteins, peptides, amino acids or nucleic acids can be coupled to CNBr-activated Sepharose, under mild conditions, via primary amino groups or similar nucleophilic groups. The activated groups react with primary amino groups on the ligand to form isourea linkages (Figure 60). The coupling reaction is spontaneous and requires no special chemicals or equipment. The resulting multi-point attachment ensures that the ligand does not hydrolyze from the matrix. The activation procedure also cross-links Sepharose and thus enhances its chemical stability, offering considerable flexibility in the choice of elution conditions. NH OH CNBr RNH O C 2 NHR HO Sepharose Sepharose OH isourea Fig. 60. Activation by cyanogen bromide and coupling to the activated matrix. 108
Options Product Spacer Coupling conditions Maximum Comments arm operating flow CNBr-activated None pH 7–9, 2–16 hours, 400 cm/h* Supplied as a freeze- Sepharose 4 Fast Flow +4 °C - room temp. dried powder. CNBr-activated None pH 8–10, 2–16 hours, 75 cm/h* Supplied as a freeze- Sepharose 4B +4 °C - room temp. dried powder. *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. There are many examples in the literature of the use of CNBr-activated Sepharose. Figure 61 shows the separation of a native outer envelope glycoprotein, gp120, from HIV-1 infected T-cells. Galanthus nivalis agglutinin (GNA), a lectin from the snowdrop bulb, was coupled to CNBr-activated Sepharose 4 Fast Flow to create a suitable affinity medium. A 280 nm 0.5 native gp120 0 24 hours 40 min Time Fig. 61. Separation of native gp120 protein on GNA coupled to CNBr-activated Sepharose 4 Fast Flow. From Gilljam, G. et al., Purification of native gp120 from HIV-1 infected T-cells. Poster presented at Recovery of Biological Products VII, Sept. 25-30, 1994, San Diego, CA, USA. Further details are available in the CNBr-activated Sepharose 4 Fast Flow datafile, from Amersham Pharmacia Biotech. Buffer preparation Acidification solution: 1 mM HCl (kept on ice) Coupling buffer: 0.2 M NaHCO 3 , 0.5 M NaCl, pH 8.3 Blocking buffer: 1 M ethanolamine or 0.2 M glycine, pH 8.0 Wash buffer: 0.1 M acetate, 0.5 M NaCl, pH 4 Preparation of CNBr-activated Sepharose 4 Fast Flow and CNBr-activated Sepharose 4B 1. Suspend the required amount of freeze-dried powder in ice-cold 1 mM HCl (HCl preserves the activity of the reactive groups that hydrolyze at high pH). 2. Wash for 15 min. on a sintered glass filter (porosity G3), using a total of 200 ml 1 mM HCl per gram dry powder, added and sucked off in several aliquots. The final aliquot of 1 mM HCl is sucked off until cracks appear in the cake. 3. Transfer the matrix immediately to the ligand solution. 109
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 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: Ligand and column preparation 1. Di
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 ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?