Affinity Chromatography: Principles and Methods by PHARMACIA FINE CHEMICALS

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0 (saliva) Purification options Binding capacity Maximum operating flow Comments HiTrap Benzamidine FF (high sub) Trypsin, > 35 mg/column Trypsin, > 175 mg/column 4 ml/min (1 ml column) 15 ml/min (5 ml column) Prepacked columns**. Benzamidine Sepharose 4 Fast Flow (high sub) Trypsin, > 35 mg/ml medium 300 cm/h* Supplied as a suspension ready for column packing**. *See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. d. of 5 cm. 05 M acetate, pH 4 containing 20% ethanol. Purification examples Figure 31 shows an example of the removal of trypsin-like proteases from human plasma to prevent proteolysis of the plasma components, using a low pH elution.

Coli, using Protein G Sepharose 4 Fast Flow. Chimeric, non-immunogenic "humanized" mouse Fab, Fab' and F(ab')2 fragments are of great interest in tumour therapy since they penetrate tumours more rapidly and are also cleared from the circulation more rapidly than full size antibodies. 5 Sample: Recombinant Fab fragment from E. coli. 0 ml Fig. 12. Purification of recombinant Fab fragments directed to the envelope protein gp120 of HIV-1 (anti-gp120 Fab), expressed in E. coli. 0 Centrifuge samples (10 000 g for 10 minutes) to remove cells and debris.

GSTrap FF 5 ml 50–60 mg recombinant GST/column 15 ml/min Prepacked column, ready to use. GSTPrep FF 16/10 >200 mg recombinant GST/column >10 ml/min Prepacked column, ready to use. Glutathione Sepharose 4 Fast Flow 10–12 mg recombinant GST/ml medium 450 cm/h* For packing high performance columns for use with purification systems and scaling up. Glutathione Sepharose 4B 8 mg horse liver GST/ml medium 75 cm/h* For packing small columns and other formats. *See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate.

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