158
2. Incubate the transformed E. coli on a LB medium containing
antibiotics (50 μg/mL kanamycin) (LBK) and 1.5% agar at
37 °C for 24 h.
3. Pick one colony up and culture it in 4 mL LBK medium at
37 °C.
4. Inoculate the 1 mL of cultured cells to 250 mL of LBK medium
in a 1000 mL flask, and culture in 37 °C.
5. When the OD 600 reaches 0.4–0.6, add to a final concentration
of 0.4 mM of IPTG, and culture the cells at 16 °C overnight.
6. Centrifuge at 10,000 × g for 15 min at 4 °C to collect the cells
(see Note 2).
7. Resuspend the pellet with 30 mL of TALON extraction buffer
and sonicate the E. coli on ice using a supersonic homogenizer
VP-15S for 2 min (50% interval) three times.
8. Centrifuge the sonicated solution at 10,000 × g for 60 min at
4 °C (see Note 3).
9. Resuspend the pellet with 25 mL of distilled water containing
1% Triton-X100 and 1 mM EDTA.
10. Centrifuge at 75,000 × g for 15 min at 4 °C. Resuspend pellet
with 25 mL of distilled water containing 1% Triton-X100 and
1 mM EDTA.
11. Repeat step 11 to wash the pellet.
12. Resuspend the pellet with 10 mL of TALON extraction buffer
containing 6 M guanidium hydrochloride (Gdn-HCl) to
solubilize it.
13. Centrifuge at 75,000 × g for 15 min at 4 °C and collect the
supernatant carefully.
14. Add 200 μL of TALON Metal Affinity Resin to the supernatant,
and rotate or shake gently for 20 min (see Note 4).
15. Centrifuge at 700 × g for 2 min, and resuspend the resin with
10 mL of TALON extraction buffer containing 6 M
Gdn-HCl.
16. Rotate or shake gently for 10 min.
17. Repeat steps 11 and 12 twice.
18. Centrifuge at 700 × g for 2 min, and collect the resin.
19. Suspend the resin with 3 mL of TALON extraction buffer containing 6 M Gdn-HCl, and put the suspension into a TALON
2-mL Disposable Gravity Column.
20. Allow to drain until it reaches the top of the resin bed.
21. Wash the column with 2 mL of TALON extraction buffer
containing 6 M Gdn-HCl.
22. Elute the protein with 1.5 mL of TALON elution buffer
containing 5.4 M Gdn-HCl.
Hiroto Iwai et al.
2. Incubate the transformed E. coli on a LB medium containing
antibiotics (50 μg/mL kanamycin) (LBK) and 1.5% agar at
37 °C for 24 h.
3. Pick one colony up and culture it in 4 mL LBK medium at
37 °C.
4. Inoculate the 1 mL of cultured cells to 250 mL of LBK medium
in a 1000 mL flask, and culture in 37 °C.
5. When the OD 600 reaches 0.4–0.6, add to a final concentration
of 0.4 mM of IPTG, and culture the cells at 16 °C overnight.
6. Centrifuge at 10,000 × g for 15 min at 4 °C to collect the cells
(see Note 2).
7. Resuspend the pellet with 30 mL of TALON extraction buffer
and sonicate the E. coli on ice using a supersonic homogenizer
VP-15S for 2 min (50% interval) three times.
8. Centrifuge the sonicated solution at 10,000 × g for 60 min at
4 °C (see Note 3).
9. Resuspend the pellet with 25 mL of distilled water containing
1% Triton-X100 and 1 mM EDTA.
10. Centrifuge at 75,000 × g for 15 min at 4 °C. Resuspend pellet
with 25 mL of distilled water containing 1% Triton-X100 and
1 mM EDTA.
11. Repeat step 11 to wash the pellet.
12. Resuspend the pellet with 10 mL of TALON extraction buffer
containing 6 M guanidium hydrochloride (Gdn-HCl) to
solubilize it.
13. Centrifuge at 75,000 × g for 15 min at 4 °C and collect the
supernatant carefully.
14. Add 200 μL of TALON Metal Affinity Resin to the supernatant,
and rotate or shake gently for 20 min (see Note 4).
15. Centrifuge at 700 × g for 2 min, and resuspend the resin with
10 mL of TALON extraction buffer containing 6 M
Gdn-HCl.
16. Rotate or shake gently for 10 min.
17. Repeat steps 11 and 12 twice.
18. Centrifuge at 700 × g for 2 min, and collect the resin.
19. Suspend the resin with 3 mL of TALON extraction buffer containing 6 M Gdn-HCl, and put the suspension into a TALON
2-mL Disposable Gravity Column.
20. Allow to drain until it reaches the top of the resin bed.
21. Wash the column with 2 mL of TALON extraction buffer
containing 6 M Gdn-HCl.
22. Elute the protein with 1.5 mL of TALON elution buffer
containing 5.4 M Gdn-HCl.
Hiroto Iwai et al.