Biology Reference
In-Depth Information
Phase mixture
& microsomes
Phase
mixture
Phase
system
Upper phase
:
RSO-PM vesicles
PEG
K
+
Cl
-
mix
spin
mix
spin
etc.
Dextran
PO
4
-
Lower phase
:
ISO PM vesicles,
tonoplast, Golgi,
peroxisomes, ER,
plastides,
mitochondria etc.
Tonoplast
sucrose
step gradient
ICM
Fig.
1
Membrane fractionation. Aqueous solutions of polyethylene glycol (PEG 3350) and Dextran T-500 sepa-
rate in two phases in the cold [
16
]. Density of Dextran is higher compared to PEG. Phosphate has a higher
affi nity for Dextran, whereas potassium is enriched in the PEG phase. Chloride has a higher affi nity for PEG
compared to potassium. Thus the hydrophobic PEG phase becomes positive and the hydrophilic Dextran phase
becomes negative. After addition of the microsomal fraction to the phase mixture, the phase system is com-
plete and reaches its fi nal concentrations. The system is mixed and separated after centrifugation. The upper
phase is transferred to a fresh lower phase, mixed and centrifugated. This step can be repeated 1-5 times.
Right-side-out (RSO) plasma membranes (PM) with a negative surface charge are enriched in the PEG phase,
whereas inside-out (ISO) PM vesicles, endoplasmatic reticulum (ER), tonoplast, Golgi vesicles, peroxisomes,
plastids, and mitochondria are enriched in the fi rst lower phase. After a washing step, the pellet of the lower
phase is applied onto a sucrose step gradient to separate the tonoplast from other intracellular membranes
(ICM) by discontinuous sucrose density centrifugation
repeated fi ve times, always with a new lower phase. Keep the
fi rst lower phase for the tonoplast preparation (explained in
Subheading
3.4
).
4. The last upper phase (PM) is washed twice with resuspension
medium (Subheading
2.1
) and pelleted both times by ultra-
centrifugation at 100,000 ×
g
for 45 min (supernatant is dis-
carded). The fi nal pellet is resuspended in resuspension medium
and stored below −70 °C until further use.
3.4
Tonoplast
1. The resulting fi rst lower phase of the plasma membrane prepa-
ration is diluted with 200 mL resuspension medium
(Subheading
2.1
), pelleted for 1 h at 50,000 ×
g
, resuspended
and washed with 200 mL resuspension medium followed by
pelletation for 30 min at 50,000 ×
g
.
2. Discard the supernatant and resuspend the pellet in 27 mL
tonoplast resuspension medium (Subheading
2.4
).
