Biomedical Engineering Reference
In-Depth Information
4. Load the 10
L of supernatants into a continuous
polyacrylamide-gradient gel (
see
Note 10
).
ʼ
1. Transfer the proteins to a nitrocellulose membrane.
2. Block the membrane by immersing into Blocking One solu-
tion for 30 min with continuous shaking on a seesaw-motion
shaker.
3. Pack the membrane in a Hybri-Bag.
4. Add 3 mL of the Can Get Signal
®
Immunoreaction Enhancer
Solution A and 3
3.7 Western Blotting
L of the anti-Pgi1p antibody to the mem-
brane. Seal the Hybri-Bag (
see
Note 11
) and mix well.
5. Incubate the packed membrane on a seesaw-motion shaker for
1.5 h at r.t.
6. Discard the solution. Transfer the membrane to a plastic tray.
7. Quickly rinse the membrane three times with PBST.
8. Wash the membrane by immersing into PBST for 5 min with
continuous shaking on a seesaw-motion shaker (r.t.).
9. Discard the solution.
10. Repeat
steps 8
and
9
two more times.
11. Pack the membrane in a new Hybri-Bag.
12. Add 3 mL of the Can Get Signal
®
Immunoreaction Enhancer
Solution B and 3
ʼ
L of anti-Rabbit antibody conjugated with
HRP to the membrane. Seal the Hybri-Bag and mix well.
13. Incubate the packed membrane on a seesaw-motion shaker for
1 h at r.t.
14. Discard the solution. Transfer the membrane to a plastic tray.
15. Quickly rinse the membrane three times with PBST.
16. Wash the membrane by immersing into PBST for 5 min with
continuous shaking on a seesaw-motion shaker (r.t.).
17. Discard the solution.
18. Repeat
steps 16
and
17
two more times.
19. Detect the proteins using ECL-Plus (or other suitable) detec-
tion reagents and ImageQuant LAS 4000 minisystem by fol-
lowing the manufacturer's instructions (
see
Note 12
).
20. After detecting Pgi1p proteins, rinse the membrane twice with
PBST.
21. Strip the antibodies by immersing the membrane into a strip-
ping solution with continuous shaking on a seesaw-motion
shaker (r.t., overnight) (
see
Note 13
).
22. Rinse the membrane three times with PBST.
23. Block the membrane (
see
step 2
).
ʼ
