Affinity Chromatography - Department of Molecular and Cellular ...

More documents

Recommendations

Info

In Activated Thiol-Sepharose 4B the hydrophilic glutathione residue acts as a spacer group thereby decreasing steric interference with exchange reactions at the terminal thiol group. The partial structure is shown in Figure 52. S epharose N CH (CH 2) 2 C NH CH CH2 S S COOH O CO NHCH 2 COOH N Fig. 52. Partial structure of Activated Thiol Sepharose 4B. In Thiopropyl Sepharose 6B the 2-hydroxypropyl residue acts as a hydrophilic spacer group. The partial structure of Thiopropyl Sepharose 6B is shown in Figure 53. S epharose O CH2 CH CH2 S S OH N Fig. 53. Partial structure of Thiopropyl Sepharose 6B. Purification options Binding capacity/ml Coupling conditions Maximum Comments medium operating flow Activated Thiol Mercaptalbumin, pH 4–8, 3–16 hours, 75 cm/h* Low capacity derivative Sepharose 4B 2–3 mg +4 °C - room temp. suitable for coupling of high molecular weight substances. Supplied as dry powder, rehydration required. Thiopropyl Ceruloplasmin, pH 4–8, 3–16 hours, 75 cm/h* High capacity derivative Sepharose 6B 14 mg +4 °C - room temp. suitable for coupling of low molecular weight substances. Supplied as dry powder, rehydration required. *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. Both media react spontaneously and reversibly under mild reducing conditions or in the presence of denaturing agents with substances containing thiol groups. 92
Performing a separation Binding buffer: 20 mM Tris-HCl, 0.1–0.5 M NaCl, pH 7.0. If required, include 8 M urea or 6 M guanidine HCl to ensure that the protein is denatured and all thiol groups are accessible for the reaction. 1 mM EDTA can be added to remove trace amounts of catalytic heavy metals. Elution buffer alternatives: For covalently bound proteins: 0.025 M cysteine, 50 mM Tris-HCl, pH 7–8. To minimize reduction of intramolecular disulphide bridges: 5–20 mM L-cysteine, 50 mM Tris-HCl, 1 mM EDTA, pH 8.0 or 20–50 mM 2-mercaptoethanol, 50 mM Tris-HCl, 1 mM EDTA, pH 8.0. Note: When using Thiopropyl Sepharose, 2-thiopyridyl groups must be removed after the protein has bound. Wash the column with sodium acetate 0.1 M, 2-mercaptoethanol 5 mM, pH 4.0 before beginning elution. N.B. Degas all buffers to avoid oxidation of free thiol groups. If the proteins to be purified contain disulphide bonds, the disulphide bridges must be reduced, for example with 2-mercaptoethanol, (5 mM). Analyse the thiol content of the sample by thiol titration to ensure that the capacity of the medium will not be exceeded. Use preliminary titration studies with 2,2'-dipyridyl disulphide to provide a guide to optimal coupling conditions. A spectrophotometer can be used to determine the release of 2-thiopyridone (absorbance coefficient = 8.08 x 10 3 M -1 cm -1 at 343 nm) when the sample (1–5 mg in 1–3 ml binding buffer) reacts with 2, 2'-dipyridyl disulphide. Choose the conditions to suit the specific sample. Under standard conditions at pH 7.5, a few minutes is usually enough for a complete reaction. 1. Use a desalting column to transfer pre-dissolved sample into the binding buffer (see page 134) and to remove any low molecular weight thiol compounds and reducing agents that might interfere with the coupling reaction. 2. Weigh out the required amount of powder (1 g gives about 3 ml for Activated Thiol Sepharose 4B and 4 ml for Thiopropyl Sepharose 6B). 3. Wash and re-swell on a sintered glass filter (porosity G3), using degassed, distilled water or binding buffer (200 ml/g, 15 min at room temperature) to remove additives. 4. Prepare the slurry with binding buffer in a ratio of 75% settled medium to 25% buffer. 5. Pack the column (see Appendix 3) and equilibrate with binding buffer. 6. Load the sample at a low flow (5–10 cm/h) and leave in contact with the medium for at least one hour to ensure maximum binding. 7. Wash the column with binding buffer until no material appears in the eluent (monitored by UV absorption at A 280 nm ). 8. Elute the target molecules with elution buffer using a low flow (5–10 cm/h). The coupling reaction can be monitored and, in some cases, quantified by following the appearance of 2-thiopyridone in the eluent at 343 nm during the purification. Sodium phosphate or ammonium acetate can be used as an alternative to Tris-HCl. Resolve different thiol proteins by sequential elution: 5–25 mM L-cysteine < 0.05 M glutathione < 0.02–0.05 M 2-mercaptoethanol < and 0.02–0.05 M dithiothreitol in 50 mM Tris- HCl, 1 mM EDTA, pH 7–8. 93
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: Media characteristics Composition M
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 ...

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

Delete template?

Save as template?