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Purification of proteins: A-alphaPiotr
Electrophoresis in polyacrylamide gel (12 %) of sonicate containing A-alpha (Fig. 1.).
 Fig. 1. Sonicate containing A-alpha protein fusion
The arrow shows place of our overexpressed protein:
1. Marker
2. sonicate A-alpha with induction
Michał L.
- Supernatant containing A-alpha was mixed with NiNta bead in proportion 10:1 and placed on rotating platform for 30 min at 4°C
- Bead was spun down at 6000 RPM, 5 min at 4°C
- Supernatant was discarded and equal amount of Elution buffer 1 (Sonication buffer A suplemented with 50 mM imidazole) was placed on top of the bead
- Bead was spun down at 6000 RPM, 5 min at 4°C
- Supernatant was collected for further analysis and equal amount of Elution buffer 2 (Sonication buffer A supplemented with 100 mM imidazole) was placed on top of the bead
- The above two steps were repeated for elution buffers 3 and 4 (150 mM and 200 mM imidazole)
- All fractions obtained in this procedure were resolved on 8% SDS-PAGE gel. It turned out that optimum imidazole concentration for elution of His-tagged A protein is 150 mM
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