By GE HEalthcare
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Additional resources for affinity chromatography principles and methods
Lane 3. Lane 4. Lane 5. Lane 6. Lane 7. Lane 8. Mr Mr 97 000 66 000 45 000 30 000 20 100 14 400 97 000 66 000 45 000 30 000 20 100 14 400 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Fig. 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.
4. Wash out unbound sample with 15 column volumes of binding buffer or until no material appears in the eluent (monitored at A280). 5. Elute the IgM with 12 column volumes of elution buffer. 6. Wash the column with 7 column volumes of wash buffer. 7. Immediately re-equilibrate the column with 5 column volumes of binding buffer. 5 M) can be used instead of ammonium sulphate. 8 M ammonium sulphate. Some monoclonal IgMs may bind too tightly to the column for complete elution in binding buffer. The remaining IgM will be eluted with wash buffer, but the high content of isopropanol will cause precipitation of IgM.
Removal of precipitated proteins: 1. Fill column with 1 M NaOH and incubate for 2 hours. 2. 0. Media characteristics Composition pH stability* Mean particle size Chelating Sepharose High Performance (HiTrap Chelating HP) Iminodiacetic acid 23 µmoles Cu2+/ml coupled to Sepharose High Performance via an ether bond. Metal ion capacity Short term 2–14 Long term 3–13 34 µm Chelating Sepharose Fast Flow Iminodiacetic acid 22–30 µmoles Zn2+/ml coupled Sepharose Fast Flow via a spacer arm using epoxy coupling.
affinity chromatography principles and methods by GE HEalthcare