Each 25 g sample was homogenized in 50 mL of chilled extraction buffer containing 50 mM Hepes (pH 7.5), 500 mM sucrose, 1 mM DTT, 5 mM ascorbate and 1% polyvinylpolypyrrolidone (PVPP).
The pellet was washed twice with chilled washing buffer (10 mM phosphate buffer, 2 mM EDTA, pH 7.5) and suspended in chilled extraction buffer (100 mM phosphate buffer, 2 mM MgCl2, 1 mM dithiothreitol, pH 7.5).
Roots were cut into approximately 5 cm segments, placed inside tubes, washed with chilled deionized water as rapidly as possible and then submerged into the chilled extraction buffer (100 mM Tris-HCl pH 7.5, 0.2 M KCl, 1 mM PMSF,) in Beckmann centrifuge bottles.
The pellet was resuspend in 300 μl of chilled Extraction Buffer 3 (1.7 M Sucrose, 10 mM Tris, pH 8, 2 mM MgCl2, 0.15% Triton X-100, 5 mM β-mercaptoethanol) and overlaid on top of 300 μl of chilled Extraction Buffer 3 and spun for 1 hour at 14,000 g at 4°C.
The cells were collected by centrifugation and disrupted by a 4 × 30 s treatment with a sonicator (Bandelin UW2070, Berlin, Germany) in chilled extraction buffer (50 mM Tris HCl, pH 7.5, with 5 mM dithiothreitol and 10% (v/v) glycerol).
Four grams of leaf tissue was ground in liquid nitrogen and vortexed in 25 ml chilled extraction buffer (0.35 M sorbitol, 0.1 M Tris HCl, 5 mM ethylenediaminetetraacetic acid [EDTA], pH 7.5, 20 mM Na2S2O5).
Pre-chilled extraction phosphate buffer (4 °C, 100 mM, 15 mL, pH 7.4) was added to the leaves with sand and poly vinylpolypyrrolidone) (PVPP) and further grinding was performed.
After washing with pre-chilled extraction buffer containing 140 mM NaCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 (pH 7.5), the bound tagged protein was eluted with 10 mM soluble glutathione dissolved in 50 mM Tris-HCl (pH 8.0), collecting 1 ml fractions for analysis by 15% SDS-PAGE and Western blotting.
The leaves of the control and NaCl treated plants were homogenized separately in chilled enzyme extraction buffer (100 mM Tris-HCl, pH 7.8 containing 10 mM MgCl2, 1 mM PMSF, 0.1 mM EDTA, 2% PVPP, 1% protease inhibitor cocktail and 10 mM DTT) in a cold room using pre-chilled mortar and pestles [ 20, 43].
Seeds were homogenized using previously cooled mortar and pestle with liquid nitrogen and extracted in a pre-chilled methanol chloroform:water extraction solution (1 2.5 1 v/v) for 30 min at 4°C shaking.
The frozen powder was weighed (70 mg), and metabolites were extracted in a 1 ml pre-chilled methanol chloroform:water extraction solution (2.5 1 1 v/v).
