By Amersham Biosciences
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Extra info for Affinity Chromatography
500 cm/h** For fast processing of large sample volumes. Retains high binding capacity at high flow rates. Supplied as a suspension ready for column packing. MabSelect™ Human IgG, approx. 30 mg/ml medium (recombinant protein A ligand) *Protein A Sepharose 4 Fast Flow and rProtein A Sepharose Fast Flow have a higher binding capacity, a more rigid matrix and provide more convenient alternatives to Protein A Sepharose CL-4B, which must be rehydrated before column packing. **See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate.
43 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. 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.
15. SDS-PAGE on PhastSystem using PhastGel 10–15, non-reduced, and silver staining. 31 Performing a separation Column: HiTrap Protein G HP, 1 ml Recommended flow rate: 1 ml/min Binding buffer: Dilute buffer concentrate 10-fold Elution buffer: Dilute buffer concentrate 10-fold Neutralization buffer: Add 60–200 µl of neutralization buffer per ml fraction to the test tubes in which IgG will be collected Centrifuge samples (10 000 g for 10 minutes) to remove cells and debris. 45 µm filter. If required, adjust sample conditions to the pH and ionic strength of the binding buffer either by buffer exchange on a desalting column (see page 133) or by dilution and pH adjustment.