Purification of proteins: A-alpha

Piotr

Electrophoresis in polyacrylamide gel (12 %) of sonicate containing A-alpha (Fig. 1.)

Purification of A-alpha with His-tag

Michał L.

1. Supernatant containing A-alpha was mixed with NiNta bead in proportion 10:1 and placed on rotating platform for 30 min at 4°C
2. Bead was spun down at 6000 RPM, 5 min at 4°C
3. 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
4. Bead was spun down at 6000 RPM, 5 min at 4°C
5. 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
6. The above two steps were repeated for elution buffers 3 and 4 (150 mM and 200 mM imidazole)
7. 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