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0 System: GSTrap FF 1 ml 10 ml extract from E. 9 mg GST-DemA 80 Elution buffer 1400 1200 1000 Column: Sample: Binding buffer: Elution buffer: Wash 800 60 Flow: 600 40 Chromatographic procedure: 400 20 200 0 ml 0 0 50 100 150 System: GSTrap FF 5 ml 50 ml extract from E. 0 mg GST-DemA 1000 Wash 800 600 Column: Sample: 400 20 200 0 0 100 0 200 300 400 500 System: GSTPrep FF 16/10 200 ml extract from E. 5% gel, Coomassie Blue staining Lane Lane Lane GST-DemA Lane Lane Lane Lane Lane Lane Lane Mr 97 000 66 000 45 000 30 000 20 100 14 400 1 2 3 4 5 6 7 8 9 10 Fig.

46 Manually using a syringe: • Sample loading • Gua-HCl wash • Urea wash 0 10 20 30 40 50 60 65 ml Lane 1. Low Molecular Weight Calibration Kit (LMW) Lane 2. Starting material for Lane 1. LMW HiTrap Chelating HP, 1 ml Lane 2. Fraction 38 Mr Lane 3. Fraction 1 Gua-HCl Lane 3. Fraction 39 wash (manually) Lane 4. Fraction 40 97 000 Lane 4. Fraction 2 Gua-HCl 66 000 Lane 5. Fraction 41 wash (manually) 45 000 Lane 6. Fraction 42 Lane 5. Fraction 3 Gua-HCl 30 000 Lane 7. Fraction 46 wash (manually) 20 100 Lane 8.

HisTrap Kit 12 mg*/column 4 ml/min As above, but includes buffers for up to 12 purifications using a syringe. 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. Chelating Sepharose Fast Flow 12 mg*/ml medium 400 cm/h** Supplied as suspension for packing columns and scale up. *Estimate for a (His)6 fusion protein of Mr 27 600, binding capacity varies according to specific protein. **See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate.

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Affinity Chromatography by Amersham Biosciences


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