By Amersham Biosciences
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Extra resources for Affinity Chromatography
HiTrap Chelating HP columns (1 ml or 5 ml) can be used with a syringe, a peristaltic pump or a chromatography system. For even larger capacity, pack Chelating Sepharose Fast Flow into a suitable column (see Appendix 3). The loss of metal ions is more pronounced at lower pH. e. all metal ions removed) between each purification if the same protein is going to be purified. e. replace metal ions) the column after 5–10 purifications. Reuse of purification columns depends on the nature of the sample and should only be considered when processing identical samples to avoid cross contamination.
5 30 000 20 100 14 400 pool I 0 5 10 Lane 1 pool II 15 20 25 30 2 3 4 ml Fig. 11. Purification of monoclonal mouse IgG1 on HiTrap Protein G HP, 1 ml. Figure 12 shows the purification of recombinant mouse Fab fragments, expressed in E. 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.
Apply the sample. 5. Wash with 10 column volumes binding buffer. 6. Wash with 2 column volumes of wash buffer or until no material appears in the eluent (determined by UV absorbance at A 280 nm). 7. * 8. 0 (the IgG may denature if left at a lower pH). 0 per ml fraction), so that the final pH of the fractions will be approximately neutral. This method, while giving a concentrated eluate, can only be used if the fusion product is stable under the acid conditions. 0. Chaotropic agents may also be used for elution.