Purification of proteins: A-alpha, Z-alpha and Z-omega

Piotr

Electrophoresis in polyacrylamide gel (12 %) of purified Z-omega and Z-alpha.

Purification of A-alpha with His-tag.

Michał L.

1. 200 ml of overnight E. coli culture was spun down 6000 RPM, 10 min at 4°C
2. The pelet 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. The post-sonication debris were spun down 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. The bead was spun down 6000 RPM, 5 min at 4°C
7. The 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. The bead was spun down 6000 RPM, 5 min at 4°C
9. The supernatant was collected for further analysis and equal amount of Elution buffer 2 (Sonication buffer A suplemented with 100 mM imidazole) was placed on top of the bead
10. Above two steps were repeated for elution buffers 3 and 4 (150mM 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 HisTagged A protein is 150 mM