Biology Reference
In-Depth Information
1. Pipette 250
L of the sample solution on a 13-cm strip holder
and place an Immobiline DryStrip (pH3-10NL, 13 cm) on it,
taking care to avoid formation of large air bubbles (
see
Note 4
).
2. Overlay the strip with 800
μ
3.2 Isoelectric
Focusing and SDS-
PAGE (2D-PAGE)
L of Cover Oil Fluid (
see
Note 5
).
3. Set and run an isoelectric focusing protocol as follows:
Dehydration, 12 h; Step 1 (step-n-hold), 500 V for 4 h; Step 2
(gradient), 1,000 V for 1 h; Step 3 (gradient), 8,000 V for
2.5 h; Step 4 (step-n-hold), 8,000 V for 1.5 h; current limit
50
μ
A/strip, run under 20 °C.
4. When the protocol is completed, pick the holders out. The
total value of V-hrs will be 22,000 or more (
see
Note 6
).
5. Pick the strip up from the holder and gently remove the oil,
pushing it to a paper towel, then place the strip into a 60-mm
dish so as to have the gel side face the inside of the dish. If not
performing the next SDS-PAGE immediately, keep the strips
stored at −80 °C until use.
6. Heat an Agarose solution tube at 100 °C on a heat-block.
7. If the strips had been kept in a freezer, place them at room
temperature until they return to RT (
see
Note 7
).
8. Add 10 mL of SDS-equilibration buffer containing 1 % DTT
to the dish and gently shake for 15 min at RT.
9. Discard the buffer and replace with 10 mL of SDS-equilibration
buffer containing 2.5 % iodoacetamide, and gently shake for
15 min at RT.
10. Pick the strip up and place it on the slot of an acrylamide gel
plate with the acidic ends to the left (
see
Note 8
).
11. Pipette slowly 1 mL of agarose solution on the strip without
creating any air bubbles between the strip and the gel. Pipette
10
μ
L of fl uorescence rainbow marker into the marker slot of
the gel before the agarose is set, and allow the gels to stand for
1-2 min until the agarose sets.
12. Fill an electrophoresis tank with 1,200 mL of SDS electropho-
resis buffer. Avoid formation of bubbles around the inner tank.
13. Run the SDS-PAGE protocol at a constant 220 V for 3 h.
14. Stop the electrophoresis when the BPB line reaches ~5 mm
from the bottom. After the electrophoresis, wash the gel in a
container fi lled with ultrapure water, followed by immersion in
blotting buffer (
see
Note 9
).
μ
3.3 Double-Stained
Immunoblotting
1. Incubate a 14 cm × 14 cm PVDF membrane in methanol at RT
for 5 min. Sandwich the gel between a fi lter paper and PVDF
membrane and place another fi lter paper on the membrane.
Roll a cylinder glass tube on the surface of the membrane
placed on the gel to remove any air bubbles. Fix them in a cassette,
