Affinity Chromatography Handbook

More documents

Recommendations

Info

44 Figure 23 shows how the purification of a GST fusion protein can be scaled up 20-fold from a GSTrap FF 1 ml column to a GSTPrep FF 16/10 column. a) GSTrap FF 1 ml A280 nm Elution buffer Elution buffer %B mAU 1200 1000 Wash 5.5 mg GST-DemA 100 80 800 60 600 400 40 200 20 0 0 0.0 10.0 20.0 30.0 ml b) GSTrap FF 5 ml A280 nm Elution buffer Elution buffer %B 1400 100 1200 1000 Wash 20.9 mg GST-DemA 80 800 60 600 400 40 200 20 0 0 0 50 100 150 ml c) GSTPrep FF 16/10 A280 nm 1400 1200 1000 800 600 400 200 0 0 Wash Elution buffer Elution buffer %B 100 111.0 mg GST-DemA 0 100 200 300 400 500 ml Electrophoresis: SDS-PAGE, 12.5% gel, Coomassie Blue staining Mr 97 000 66 000 45 000 30 000 20 100 14 400 1 2 3 4 5 6 7 8 9 10 Fig. 23. Scaling up purification of a GST fusion protein. 80 60 40 20 GST-DemA Column: GSTrap FF 1 ml Sample: 10 ml extract from E. coli expressing GST-DemA Binding buffer: PBS, pH 7.4 Elution buffer: 50 mM Tris-HCl, pH 8.0 with 10 mM reduced glutathione Flow: Sample loading: 0.5 ml/min Wash and elution: 1 ml/min Chromatographic procedure: 5 ml binding buffer, 10 ml sample, 10 ml binding buffer, 7 ml elution buffer, 5 ml binding buffer System: ÄKTAexplorer Column: GSTrap FF 5 ml Sample: 50 ml extract from E. coli expressing GST-DemA Binding buffer: PBS, pH 7.4 Elution buffer: 50 mM Tris-HCl, pH 8.0 with 10 mM reduced glutathione Flow: Sample loading: 2.5 ml/min Wash and elution: 5 ml/min Chromatographic procedure: 25 ml binding buffer, 50 ml sample, 50 ml binding buffer, 35 ml elution buffer, 25 ml binding buffer System: ÄKTAexplorer Column: GSTPrep FF 16/10 Sample: 200 ml extract from E. coli expressing GST-DemA Binding buffer: PBS, pH 7.4 Elution buffer: 50 mM Tris-HCl, pH 8.0 with 10 mM reduced glutathione Flow: Sample loading: 5 ml/min Wash and elution: 10 ml/min Chromatographic procedure: 100 ml binding buffer, 200 ml sample, 200 ml binding buffer, 140 ml elution buffer, 100 ml binding buffer System: ÄKTAexplorer Lane 1: Low Molecular Weight (LMW) calibration kit, reduced Lane 2: Extract of E. coli expressing GST-DemA, 1 g cell paste/5 ml Lane 3: Flow-through from GSTrap FF 1 ml Lane 4: GST-DemA eluted from GSTrap FF 1 ml Lane 5: Extract of E. coli expressing GST-DemA, 1 g cell paste/5 ml Lane 6: Flow-through from GSTrap FF 5 ml Lane 7: GST-DemA eluted from GSTrap FF 5 ml Lane 8: Extract of E. coli expressing GST-DemA, 1 g cell paste/5 ml Lane 9: Flow-through from GSTPrep FF 16/10 Lane 10: GST-DemA eluted from GSTPrep FF 16/10
Performing a separation Binding buffer: 140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.3 Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0 A B C Fig. 24. Using GSTrap FF with a syringe. A: Prepare buffers and sample. Remove the column’s top cap and twist off the end. B: Equilibrate column, load the sample and begin collecting fractions. C: Wash and elute, continuing to collect fractions. 1. Equilibrate the column with 5 column volumes of binding buffer. 2. Apply the sample. 3. Wash with 5–10 column volumes of binding buffer. 4. Elute with 5–10 column volumes of elution buffer. 5. Wash with 5–10 column volumes of binding buffer. It is important to keep a low flow rate during sample loading and elution as the kinetics of the binding interaction between GST and glutathione are relatively slow. The binding capacity is protein dependent and therefore yield will vary according to the type of protein. Yield may be improved by using a slower flow rate or passing the sample through the column several times. For a single purification of a small quantity of product or for high throughput screening, GST MicroSpin columns are convenient and simple to use with either centrifugation or MicroPlex 24 Vacuum. To increase capacity, connect several GSTrap FF columns (1 ml or 5 ml) in series. For greater capacity, use GSTPrep 16/10 FF columns or pack Glutathione Sepharose 4 Fast Flow into a suitable column (see Appendix 3). GSTrap FF columns can be used with a syringe, a peristaltic pump or a chromatography system. Enzyme-specific recognition sites are often included to allow the removal of the GST tag by enzymatic cleavage when required. Thrombin is commonly used for enzymatic cleavage, and must, subsequently, be removed from the recombinant product. HiTrap Benzamidine FF (high sub) 1 ml or 5 ml columns provide simple, ready-made solutions for this process (see page 54). Reuse of GSTrap FF depends on the nature of the sample. To prevent cross-contamination, columns should only be reused with identical samples. 45
Page 1 and 2: 18-1022-29 Edition AD Affinity Chro
Page 3 and 4: Affinity Chromatography Principles
Page 5 and 6: DNA binding proteins ..............
Page 7: Appendix 4 ........................
Page 10 and 11: 8 This handbook describes the role
Page 12 and 13: 10 Affinity chromatography is also
Page 14 and 15: 12 BioProcess Media for large-scale
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: A 280 nm 0.6 0.4 0.2 0 Fig. 8. Scou
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 0.0 0 200
Page 39 and 40: Desalt and/or transfer purified IgG
Page 41 and 42: Performing a separation Column: HiT
Page 43 and 44: Fig. 21. SDS-PAGE of non-reduced sa
Page 45: Purification options Binding capaci
Page 49 and 50: Poly (His) fusion proteins His Micr
Page 51 and 52: One step, on-column, refolding and
Page 53 and 54: Purification using HisTrap Kit HisT
Page 55 and 56: 1. Pack the column (see Appendix 3)
Page 57 and 58: Purification options Binding capaci
Page 59 and 60: 1. Equilibrate the column with 5 co
Page 61 and 62: Specifically bound biomolecules can
Page 65 and 66: Sample: 2000 ml partially purified
Page 67 and 68: Coagulation factors HiTrap Heparin
Page 71 and 72: Purification or removal of fibronec
Page 73 and 74: Purification examples Figure 42 sho
Page 75 and 76: Media characteristics Ligand and de
Page 77 and 78: Media characteristics Ligand densit
Page 81 and 82: Elution buffers: use low concentrat
Page 85 and 86: Lentil lectin for binding of branch
Page 89 and 90: Remove proteases as quickly as poss
Page 91 and 92: Purification example A 280 nm 1.0 0
Page 93 and 94: Samples: 2.5 ml cell extract contai
Page 95 and 96: In Activated Thiol-Sepharose 4B the
Page 97 and 98:
Reactivation Pass one to two column
Page 99 and 100:
Chapter 4 Components of an affinity
Page 101 and 102:
If several functional groups are av
Page 103 and 104:
Chapter 5 Designing affinity media
Page 105 and 106:
Coupling the ligand 1. Prepare the
Page 107 and 108:
Increase the ligand concentration t
Page 109 and 110:
Options Product Spacer arm Coupling
Page 111 and 112:
Media characteristics Product Ligan
Page 113 and 114:
Preparation of the matrix should be
Page 115 and 116:
Immunoaffinity chromatography Immun
Page 117 and 118:
Preparation of coupling reagent Use
Page 119 and 120:
Coupling through hydroxy, amino or
Page 121 and 122:
Coupled glycylleucine % Coupled lig
Page 123 and 124:
Coupling through a thiol group Thio
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:
Sample clarification Centrifugation
Page 133 and 134:
Examples of precipitation agents ar
Page 135 and 136:
Resolubilization of protein precipi
Page 137 and 138:
Alternative 1: Manual desalting wit
Page 139 and 140:
Appendix 2 Selection of purificatio
Page 141 and 142:
4. Estimate the amount of slurry (r
Page 143 and 144:
Appendix 5 Conversion data: protein
Page 145 and 146:
Middle unit residue (-H 20) Charge
Page 147 and 148:
Expected results when changing cond
Page 149 and 150:
Expected results with competitive e
Page 151 and 152:
Appendix 8 Analytical assays during
Page 153 and 154:
Appendix 9 Storage of biological sa
Page 155 and 156:
Additional reading Code No. Purific
Page 157 and 158:
Product Quantity Code No. Blue Seph
Page 159 and 160:
STREAMLINE, Sepharose, HiTrap, ÄKT
show all

Affinity Chromatography Handbook

Create successful ePaper yourself

Delete template?

Save as template?