Biology Reference
In-Depth Information
and centrifuged at 7000 rpm for 5 min at 4
C. The supernatant is collected and the
pellet is resuspended in LD-A. Spheroplast disintegration and centrifugation are
repeated with the same procedure. Both resulting supernatants are combined and trans-
ferred into an Ultra-Clear Centrifuge Tube (Beckman). Each tube is filled up to 1/3
with the supernatant which is then carefully overlaid with LD-A to the top of the tube.
Ultracentrifugation at 28,000 rpm for 45 min at 4
C using a swing out rotor yields
a white layer on top (crude LD) that can be removed with a spatula and transferred
into a 15-mL Dounce homogenizer. The crude LD are homogenized with 8 strokes
using a loose fitting pestle in the presence of 1 mM PMSF. Then, the sample is
transferred to a new ultracentrifuge tube (1/4 of the total tube volume) and carefully
overlaid with LD-B (8% Ficoll 400 in 10 mM MES/Tris [pH 6.9], 0.2 mM Na
2
ED-
TA
2H
2
O). Ultracentrifugation at 28,000 rpm for 30 min at 4
C results in a top layer
containing LD. This top layer is again removed and transferred to a 15-mL Dounce
homogenizer where the LD are homogenized with 8 strokes using a loose fitting pestle
in the presence of 1 mM PMSF. Prior to the last ultracentrifugation step, buffer LD-C
(0.25 M sorbitol in 10 mMMES/Tris [pH 6.9], 0.2 mMNa
2
EDTA
2H
2
O) is filled into
a fresh ultracentrifuge tube up to 3/4 of the tube volume. The homogenized LD are
loaded to the bottom of the tube with the aid of a syringe. The last ultracentrifuge step
at 28,000 rpm for 30 min at 4
C yields a top layer containing highly purified LD. The
top layer is collected with a pipette and transferred into a 7 mL Dounce homogenizer,
and LD are mixed with 8 strokes using a loose fitting pestle. LD can then be stored at
80
C for further analysis. If required, the pellet from the last centrifugation step con-
taining mainly vacuoles can be collected and analyzed as well. Isolation of LD from
P. pastoris
and
Y. lipolytica
can be performed employing the same protocol with minor
modifications (see Notes 2 and 3).
2.2.2
Protein analysis of LD
2.2.2.1
Protein determination
Prior to protein determination LD fractions have to be delipidated. Therefore, lipids
are extracted with two volumes of diethyl ether with repeated vigorous shaking.
After centrifugation at top speed in a table top centrifuge, the organic phase is with-
drawn and residual diethyl ether is removed under a stream of nitrogen (see Notes 4
and 5). Proteins are precipitated with TCA at a final concentration of 10% for 1 h on
ice, and the resulting pellets are solubilized in 0.1% SDS/0.1 M NaOH for protein
quantification. In a typical procedure, 200
m
LH
2
O bidest. and 100
m
L TCA
(50%) are added to 200
L of an isolated, delipidated LD fraction for precipitation.
Proteins are quantified by the method of
Lowry, Rosebrough, Farr, and Randall
(1951)
, which is suitable for the quantification of membrane proteins due to the fact
that detergents such as SDS can be included. Moreover, this method is more sensitive
than the Biuret method (
Smith et al., 1985
). Bovine serum albumin is used as a stan-
dard. The expected protein concentrations for LD fractions are between 0.01 and
0.2 mg/mL depending on culture conditions and strain background (see Note 6).
m
