Biology Reference
In-Depth Information
3. Transfer the 125
l of substrate mixture to a microtiter plate.
4. Carefully pipette the 25
μ
l of translation mixture underneath
the substrate mixture to form the bilayer reaction.
5. Seal the plate to avoid evaporation.
6. Incubate the plate at 26 °C for 16 h.
μ
1. Pour 40 g of Dowex resin into a beaker.
2. Add 400 ml of 1 N HCl and stir for 1 h at room temperature.
3.4 Preparation
of Dowex resin
3. Pour the resin suspension into Buchner funnel lined with fi lter
paper.
4. After the fl uids fi lter through, transfer the Dowex resin back
into the beaker.
5. Repeat
steps 2
-
4
with 400 ml of 1 N NaOH followed by
400 ml of 200 mM sodium acetate.
6. To the beaker of Dowex resin add 400 ml of 200 mM sodium
acetate and stir for 1 h at room temperature (
see
Note 4
).
7. Plug the end of Pasteur pipette with absorbent cotton using a
stick (
see
Note 5
).
8. Pour the Dowex resin into the Pasteur pipette. The resin bed
height should be approximately 5 cm for columns to remove
unincorporated substrates from liposomes, or 4.2 cm for trans-
port assay columns.
9. Equilibrate these resins by passing either 6 ml of equilibration
solution for columns to remove unincorporated substrates
from liposomes, or 5.5 ml of 200 mM sodium acetate for
transport assay columns.
3.5 Preparation
of Substrate-
Preloaded Liposomes
1. Suspend 80 mg of the acetone-washed phospholipid mixture
with 1 ml of 2× substrate solution.
2. Sonicate the suspension (10 % amplitude and 30 % duty cycle)
on ice until the suspension changes in appearance from milky
to nearly transparent.
1. After protein synthesis, mix the bilayer reaction mixture
(150
3.6 Preparation
of Transport Assay
Proteoliposomes
l) gently, and transfer it to a new microfuge tube.
2. Centrifuge the reaction mixture (20,000 ×
g
, 20 min, 4 °C)
(
see
Note 6
).
3. Resuspend the pellet fraction in 150
μ
μ
l of 10 mM PIPES-
NaOH, pH 6.5.
4. Sonicate the resuspended pellet at room temperature for 18 s
(10 % amplitude and 50 % duty cycle).
5. To verify expression of the target protein, analyze the resus-
pended pellet by sodium dodecyl sulfate-polyacrylamide gel
