Biology Reference
In-Depth Information
6.
Clean the slides in an argon plasma cleaner prior to vesicle fusion: Put a slide in
the plasma cleaner, close and evacuate the chamber, flush the chamber carefully
with argon, (the vacuum should be kept at low pressure), apply high voltage. The
purple color of the plasma is now visible. Leave the slide inside for 15 min.
7.
Take the slide out of the plasma cleaner and put it into the analysis chamber. Fill
the chamber with Buffer C and inject about 50 nmol of the vesicle suspension.
Stir the buffer during the fusion process, which is finished after about 30 min.
Wash the analysis chamber with Buffer C to remove excess of vesicles. The fluo-
rescence signal should be stable after the washing step (
see
Note 13
).
4. Notes
1.
Prevent DMSO from absorbing water, this will reduce the amount of active
fluorophores before the labeling reaction is started.
2.
The reconstitution was also performed using other lipids/lipid mixtures as
POPC, POPC/POPS (molar ratio 75:25), POPC/POPG (molar ratio 50:50),
PC/PE (molar ratio 70:30), which did in neither case result in a good integrin
reconstitution. With pure DMPC the result was comparable to the equimolar mix-
ture of DMPG/DMPC.
3.
When 100 m
M
octyl
-D-glucopyranoside or 30 m
M
octyl-POE were used to solubi-
lize lipid and protein no reconstitution could be seen in the electron microscope.
β
4.
Washed BIO-Beads were stored in 20% EtOH at 4
°
C and washed again with H
2
O
prior to use. The beads were not allowed to dry.
5.
Solutions of MnCl
2
are not very stable because of oxidation to MnO
2
and should
always be freshly prepared.
6.
For control measurements, vesicles without protein were prepared using the same
reconstitution protocol, but the incubation time for the first addition of BIO-Beads
was reduced to 120 min. For reconstitution of fluorescently labeled integrin the
reconstitution protocol was used with the following modifications: the integrin-
lipid mixture was incubated twice with only 25 mg of BIO-Beads for 90 min and
60 min, respectively.
7.
The supernatant can be easily removed from the beads with a plastic pipet tip that
was squeezed with a pair of tweezers, so that you can only take up the fluid, not
the beads.
8.
The precipitation can also be done at -20
°
C for 15 min.
9.
The first reaction is not an end point reaction and must, therefore, be timed accu-
rately. 15 s between tubes should be sufficient to resuspend the precipitate.
10.
The absorbance of the reference in the phosphate determination should be 0.012-
0.020. If higher, the molybdate solution was too old. The absorbance should be
about 0.1-0.125/ 20 nmol of phosphate. Using linear regression, the regression
coefficient is generally better than 0.997.
11.
Calculate the molar protein to lipid ratio, for a good preparation it should be in
between 1:1600 to 1:1200.
12.
Use only stain at neutral pH and do not stain with conventional uranyl acetate or
uranyl formiate, which would destroy the vesicles.
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