Microsomal fraction and plasma membrane isolation
A pool of leaves from five different plants of the same treatment was
used for four microsomal fraction extractions in order to obtain enough
protein and lipid amounts for further analysis. Leaves plasma membranes
were purified from microsomal fractions using the two-phase aqueous
polymer technique first described by Larsson et al. , (1987) and
modified by Casado-Vela et al. , (2010).
Leaves (50 g) were cut before vacuum infiltrated with 0.5 g of PVP and
160 ml of extraction buffer containing 0.5 M sucrose, 10% glycerol, 20
mM Na2EDTA, 20 mM EGTA, 50 mM NaF, 5 mM
β-glycerophosphate, 1 mM 1,10-phenanthroline, 1 mM
Na3VO4, 0.6% PVP, 5 mM ascorbic acid, 5
mM DTT, and 0.5 mg/L leupeptin in 50 mM Tris-MES, pH 8.0. After buffer
infiltration, tissues were homogenized using a blender and filtered
through a nylon mesh (pore diameter of 100 µm). The homogenate was
centrifuged at 10,000 x g for 40 min at 4°C. The supernatant was
collected and centrifuged at 100,000 x g for 45 min at 4°C. The
pellet was suspended in buffer (FAB) containing 330 mM sucrose, 2 mM
DTT, 10 mM NaF, and 5 mM phosphate buffer (pH 7.8). Plasma membranes
were purified from microsomal fraction by partitioning in a two
phase-system mixture with a final concentration of PEG-3350
(Sigma)/Dextran-T500 (GE Healthcare), 6.0% (w/w), in the presence of
330 mM sucrose, 5 mM KCl, 5 mM potassium phosphate (pH 7.8). The
two-phase system was centrifuged for 5 min at 4000 x g at 4°C.
The upper phase, enriched plasma membranes, was recollected and washed
in 300 mM sucrose, 9 mM KCl, 6 mM Na2EDTA, 6 mM EGTA, 60
mM NaF, and Tris-borate, pH 8.3, and centrifuged at 100,000 x g
for 45 min, 4°C. The resulting pellet was suspended in resuspension
buffer (FAB). The protein concentration of both, microsomal fraction and
plasma membrane-enriched fractions, was determined with Protein assay
dye reagent (BioRad), with BSA as standard.