By PHARMACIA FINE CHEMICALS
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19b. SDS-PAGE on PhastSystem, using PhastGel 4–15 with silver staining. 5 with 30% isopropanol The sample must have the same concentration of ammonium sulphate as the binding buffer. 8 M. Stir slowly and continuously. 45 µm filter immediately before applying it to the column. 8 M ammonium sulphate. 0 M. To avoid precipitation of IgM, it is important to add the ammonium sulphate slowly. An increased concentration of ammonium sulphate will cause more IgG to bind, which might be a problem if serum has been added to the cell culture medium.
HiTrap columns can be used with a syringe, a peristaltic pump or connected to a liquid chromatography system, such as ÄKTAprime. Reuse of HiTrap lgM Purification HP depends on the nature of the sample and should only be considered when processing identical samples to avoid cross-contamination. 39 Media characteristics HiTrap IgM Purification HP Ligand and density pH stability* 2-mercaptopyridine 2 mg/ml Long term 3–11 Short term 2–13 Mean particle size 34 µm *Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance.
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. d. of 5 cm. 47 Purification examples Figures 25 and 26 show the purification of recombinant proteins expressed in soluble form or as inclusion bodies and Figure 27 gives an example of simultaneous on-column purification and refolding of a recombinant protein expressed as an inclusion body.