Membrane Preparation
Membrane fractions were prepared from transfected HEK293 cells as
described previously (Zöllner, 2003). The cells were grown in 175 cm2
tissue culture flasks to approximately 90 % confluence. Cells were then
washed with Tris buffer (50 mM, Trizma preset crystals, pH 7.4; Sigma
Aldrich, Darmstadt, Germany), harvested with a scraper, homogenized
using a mechanical disperser (Dispergierstation T8.10, IKA-Werke,
Staufen, Germany) at maximum speed for 10 s and centrifuged at 42K x g
for 20 min at 4oC (Avanti JXN-26 ultracentrifuge, Beckmann Coulter,
Krefeld, Germany). Cellular pellets including membranes with embedded
and anchored proteins were then resuspendend in Tris buffer for washing
to separate them from cytosolic components by homogenization and
centrifugation at the same settings. Supernatants were discarded and the
pellets were stored at -80 oC. On the day of usage, the pellets were
thawed on ice in Tris buffer and homogenized. Total protein
concentrations were determined according to the Bradford method (Bio-Rad
Laboratories GmbH, München, Germany). (Bradford, 1976) and homogenates
were split according to the number of conditions tested in respective
assay buffers.