Purification of proteins: A-alpha

Piotr

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

Purification of A-alpha with His-tag

Michał L.

1. 200 ml of overnight E. coli culture was spun down at 6000 RPM, 10 min at 4°C
2. Pellet was resuspended in 20 ml of sonication buffer A (50 mM phosphate buffer pH 7.2, 100 mM NaCl, 10 mM imidazole)
3. The mixture was sonicated in 20 pulses: 90 W pulse duration - 20 s, pause 40 s.
4. Post-sonication debris were spun down at 13200 RPM, 20 min at 4°C
5. Clear supernatant was mixed with NiNta bead in proportion 10:1 and placed on rotating platform for 30 min at 4°C
6. Bead was spun down at 6000 RPM, 5 min at 4°C
7. 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
8. Bead was spun down at 6000 RPM, 5 min at 4°C
9. 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
10. The above two steps were repeated for elution buffers 3 and 4 (150 mM and 200 mM imidazole)
11. 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