Biomedical Engineering Reference
In-Depth Information
GTP, either 10
ʼ
g mixed adaptors or 1 mg cytosol, and reaction
buffer to 200
L. The contribution of amphiphysins is tested by
supplementing up to 50
ʼ
g purifi ed protein. The mixture is
incubated for 30 min at 37 °C to allow nucleotide exchange on
Arf1 and protein recruitment.
ʼ
3. The reaction is mixed in a 4-mL ultracentrifuge tube with
0.4 mL 60 % sucrose solution (to a fi nal concentration of 40 %
sucrose). The mixture is carefully overlayed with 1 mL of 20 %
sucrose solution. After removal of any foam at the top with a
pipette, 20 % sucrose solution is added to a total of 3.82 mL.
4. The tubes are balanced and 180
L assay buffer is overlayed to
facilitate liposome collection afterwards.
5. Samples are centrifuged at 300,000 ×
g
for 1 h (Kontron
TST60.4 rotor, at 55,000 rpm).
6. Four 1-mL fractions are collected from the top.
7. TCA precipitation: the fractions are mixed with 160
ʼ
L TCA
in 1.5-mL tubes and centrifuged in a microfuge at maximal
speed for 15 min.
8. To the pellets, add 850
ʼ
L ice-cold acetone, and the tubes are
centrifuged again for 5 min at 4 °C.
9. The pellets are air-dried for 15-20 min at room temperature
and dissolved in 60
ʼ
L SDS-sample buffer by pipetting up and
down and by heating at 95 °C for 10 min (
see
Note 17
).
10. Samples are separated by gel electrophoresis on a 12.5 % SDS-
gel and analyzed by immunoblotting for AP-1 (
ʼ
-adaptin),
Arf1, and amphiphysin as appropriate. Example results are
shown in Fig.
1
(dependence of AP-1 recruitment on Arf1,
lipid composition, and cargo signals), Fig.
2b
(left lanes:
cargo-independent recruitment of AP-1 using cytosol), and
Fig.
3
(stabilization of AP-1 on liposomes by different
amphiphysins).
ʳ
To analyze the oligomerization state of liposome-associated
protein, membranes in the fl oated fraction are solubilized and the
protein centrifuged into a sucrose density gradient. Protein in the
gradient fraction is detected by immunoblot analysis. The proce-
dure is based on ref.
10
.
3.7 Sedimentation
Assay
1. The top 350
L of a fl oatation gradient (Subheading
3.6
,
step 5
), which contains most of the fl oated liposomes, is col-
lected and mixed with 350
ʼ
ʼ
L assay buffer to dilute the
sucrose.
2. The lipid membranes are solubilized by addition of 0.5 %
Triton X-100.
3. The sample is loaded onto a 4.3 mL 10-25 % sucrose gradient
with 0.2 % Triton X-100 and centrifuged at 100,000 ×
g
for 5 h
(Kontron TST 55.5 rotor, at 30,000 rpm).
