Biomedical Engineering Reference
In-Depth Information
12. After 2 h, centrifuge the bead and sample mixture for 1 min
at 3,000 ×
g
to pellet beads.
13. Remove the sample supernatant with a pipette and set aside.
14. Wash the beads by resuspension in 0.5 mL of washing buffer
(
see
Note 13
).
15. Centrifuge the beads for 1 min at 3,000 ×
g
to pellet beads.
16. Remove wash buffer by vacuum aspiration (
see
Note 11
).
17. Repeat the wash step 2 more times (
steps 12
-
14
).
18. After the 3rd wash, remove wash buffer using vacuum aspira-
tion. Then, using a fi ne pipette tip, carefully remove the resid-
ual wash buffer from the beads.
19. Resuspend the beads in 45
ʼ
L of elution buffer. Boil the sam-
ple for 5 min at 95 °C.
3.4 SDS-PAGE
Electrophoresis
and Western Blot
1. Load boiled samples onto the SDS-PAGE gel and run at 150 V
until the dye front reaches the bottom of the gel.
2. When the gel is almost fi nished running, prepare the transfer
apparatus: soak sponges (2) and fi lter paper (2) in transfer buf-
fer. Soak the nitrocellulose membrane in 20 % methanol
solution.
3. After electrophoresis, pry open the glass plates and gently place
the gel onto the fi lter paper/sponge soaked in transfer buffer.
4. Gently place the nitrocellulose membrane on top of the gel
(
see
Note 14
).
5. On top of the membrane, add the second layer of the fi lter
paper and sponge that was soaked in transfer buffer.
6. Close the cassette and place into the transfer chamber.
7. Fill the chamber with transfer buffer and insert stir bar and ice
block.
8. Run the transfer at 100 V for 1 h on top of a stir plate running
at maximum speed.
9. After the transfer, trim the membrane and place inside a clean
blotting box with PBS (
see
Note 15
).
10. Block the membrane for 30 min using Odyssey blocking buffer
containing 0.01 % Tween. Incubate the membrane at room
temperature on a table rocker (
see
Note 16
).
11. After blocking, add primary antibodies (Myc: 1:10,000, GFP:
1:2,000) and incubate for 1 h while rocking at RT.
12. After the primary antibody incubation, wash the membrane
4 times by adding 5-10 mL of PBS and rocking for 5 min.
13. After washing, add the secondary antibody solution diluted in
PBS containing 5 % milk (both secondary antibodies diluted
