Pharmalyte ph 3 10

Proteins were isolated from 0.5 g fresh weight of calli using a phenol extraction methanol/ammonium acetate precipitation method^{29 (link)}. The phenolic phase was collected and precipitated overnight in five volumes of 100 mM ammonium acetate in ethanol at −20 °C. After centrifugation (10 min, 6000 g, 4 °C), the pellet was washed twice with ice-cold acetone.
For isoelectric focusing, dried protein pellets were dissolved in IPG buffer (9.5 M urea, 4% w/v CHAPS, 2% Pharmalyte pH 3–10 (GE Healthcare, Uppsala, Sweden), DeStreak Reagent (GE Healthcare) and 0.01% w/v bromophenol blue). Protein concentration was determined using an RC/DC kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). A total of 500 µg of whole protein sample in 350 ml IPG buffer was applied to 18-cm Immobiline DryStrip pH 3–10 NL (GE Healthcare) by passive rehydration for 12 h at 20 °C according to the manufacturer’s recommendations. IEF was performed in a Protean IEF Cell (Bio-Rad) for 60,000 V-h. Before separation in the second dimension, the Immobiline DryStrip was equilibrated in buffer (6 M urea, 0.375 M Tris-HCl, pH 8.8, 2% SDS, 20% glycerol, and 2% DTT) for 10 min. For SDS-PAGE, 12% polyacrylamide gels with 4% stacking gels were run in a Protean II xi cell (Bio-Rad). The gels were stained with Coomassie Brilliant Blue G-250. Three control and three experimental gels were used in the analysis.

Reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise noted. Proteins were isolated from the fresh biomass of Va and VaCa calli (1 g fresh weight) using a SDS extraction buffer and a cold 20% TCA/acetone precipitation method. Protein quantification was performed using the Bradford assay. For isoelectric focusing, dried protein pellets were dissolved in the IPG buffer, containing 9.5 M urea with thiourea, 4% w/v CHAPS, 65 mM DTT, 2% Pharmalyte pH 3–10 (GE Healthcare, Uppsala, Sweden), and 0.01% w/v bromophenol blue. A protein probe diluted in the IPG buffer was loaded to a 11-cm Immobiline DryStrip pH 3–10 NL (GE Healthcare, Uppsala, Sweden) according to the manufacturer’s recommendations by passive rehydration for 12 h at 20 °C. IEF was performed in a Protean IEF Cell (Bio-Rad Laboratories Inc., Hercules, CA, USA) for 60,000 V-h. For SDS-PAGE, 12% polyacrylamide gels with 4% stacking gels were run in a Protean II xi cell (Bio-Rad Laboratories Inc., Hercules, CA, USA). The gels were stained with Coomassie Brilliant Blue G-250. A set of three gels for Va and VaCa was used in the analysis.