Equilibration buffers 1 2

Total protein was isolated from 1.5 g of leaf tissue using phenol extraction method. In brief, the tissue was ground in liquid nitrogen and suspended in extraction buffer (700 mM Sucrose, 500 mM Tris–HCl, pH7.5, 50 mM EDTA, 100 mM KCl, 2% (w/v) β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride) and protein extraction was done following standard protocol. The isolated protein was resuspended in IEF buffer consisting of 7 M urea, 2 M thiourea, 4% 3-[(3-cholamido propyl)-dimethylammonio]- 1-propane sulfonate (CHAPS), 20 mM DTT and 1% (w/v) Bio-Lyte (3/10) ampholyte (BioRad Laboratories, Hercules,CA, USA) as standardized before [35 (link),36 (link)]. The protein concentration was determined by Bradford’s method [37 (link)]. 100 μg of protein was used to passively re-hydrate immobilized pH gradient strip (7 cm; pH4–7; BioRad Laboratories, Hercules, CA, USA) for 12 h. IEF was performed as follows: 250 V for 30 min, 4000 V for 2 h, 4000 V for 10000 V-h, 500 V for 1 h on BioRad PROTEAN IEF Cell system (BioRad Laboratories, Hercules, CA, USA). Focused strips were then equilibrated in equilibration buffers I & II (BioRad Laboratories, Hercules, CA, USA) for 15 min each. For running gels in the second dimension, 12% SDS polyacrylamide gels were used and stained with colloidal Coomasie Brilliant Blue (CBB) G-250 [38 (link)].

About 2 gm of leaf tissue of combined stress treated Col-0, ein2 and aba1.6 was used for total protein extraction by phenol extraction method. The tissue was then finely ground in liquid nitrogen and suspended in extraction buffer (700 mM Sucrose, 500 mM Tris–HCl, pH7.5, 50 mM EDTA, 100 mM KCl, 2% (w/v) β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride) and protein extraction was done following standard protocol. The isolated protein was further resuspended in IEF buffer consisting of 7 M urea, 2 M thiourea, 4% 3-[(3-cholamido propyl)-dimethylammonio]- 1-propane sulfonate (CHAPS), 20 mM DTT and 1% (w/v) Bio-Lyte (3/10) ampholyte (BioRad Laboratories, Hercules, CA, USA) as standardized before. Bradford’s method was used for the measurement of protein concentration. 800 μg of protein was passively re-hydrated in immobilized pH gradient strip (11 cm; pH4–7; BioRad) for 12 h. IEF was performed as follows: 250 V for 30 min, 8000 V for 2 h, 8000 V for 26000 V-h, 750 V for 1 h on BioRad PROTEAN IEF Cell system (BioRad). Focused strips were further equilibrated in equilibration buffers I & II (BioRad) for 15 min each. Second dimension gels were run by using 12% SDS polyacrylamide gels and stained with colloidal Coomasie Brilliant Blue (CBB) G-250.