Biomedical Engineering Reference
In-Depth Information
buffer containing 100, 150, 200, and 250 mM imidazole.
Collect 1-mL fractions and determine protein concentration.
10. The fraction with the highest protein concentrations are col-
lected, snap-frozen in 30-
ʼ
g aliquots, and stored at −80 °C.
3.5 Preparation
of Liposomes
and Peptidoliposomes
Liposomes are prepared by extrusion through a defi ned pore-size
fi lter. A maleimide-containing lipid is included to allow chemical
coupling of cysteine-terminal peptides. The procedure is based on
refs.
7
and
20
.
1. Five micromoles of the desired lipids, for example 3.8 mg egg
PC, soybean lipids, or a 1:1 mixture of the two, is dissolved in
1 mL chloroform/methanol in a test tube that can stand liquid
nitrogen.
2. To produce peptidoliposomes, 125 nmol (2.5 mol %) MMCC-
DOPE is dissolved in 100
L chloroform/methanol and added
to the lipids (
see
Note 11
).
3. The organic solvent is evaporated under a stream of nitrogen.
4. The lipids are redissolved in 2 mL of 1,1-dichloromethane and
dried again under nitrogen.
5. Meanwhile, the extruder is assembled and washed with lipo-
some buffer, and 0.28
ʼ
g) of the peptides to be
coupled (approximately fourfold excess over the reactive lipid,
assuming half of it is exposed to the outside of the liposome) is
weighed out and dissolved in 100
ʼ
mol (400
ʼ
L liposome buffer.
6. The dried lipids are suspended in 1 mL liposome buffer by fi ve
cycles of vortexing, shock-freezing in liquid nitrogen, and
thawing under warm tap water (
see
Note 12
).
7. Lipids are passed 11 times through the extruder unit (
see
Note 13
).
8. To couple peptides to the liposomes, they are immediately
mixed with 100
ʼ
L 4 mg/mL peptide and incubated for 1 h at
room temperature (
see
Note 14
).
9. The fi nal peptidoliposomes are stored at 4 °C with 0.02 %
(w/v) NaN
3
for up to 2 weeks (
see
Notes 15
and
16
).
ʼ
3.6 Floatation Assay
Peptidoliposomes are incubated with coat proteins, Arf1, and
nucleotides to allow for Arf1 activation and coat recruitment, and
then fl oated on a sucrose step gradient. Proteins associated with
the liposomes or remaining in the loading zone are collected by
trichloroacetic acid (TCA) precipitation and detected by immu-
noblot analysis. The procedure is based on refs.
7
and
10
.
1. Cytosol is centrifuged at 170,000 ×
g
for 30 min (Beckman
TLA100.3 rotor, 85,000 rpm) to remove aggregates.
2. Liposomes or peptidoliposomes (100
ʼ
L containing
∼
0.5
ʼ
mol
lipid) are mixed with 5
ʼ
g Arf1, 0.2 mM GMP-PNP or 2 mM
