Protean ief unit

Proteomic profiling and identification in limonene treated E. coli was performed using 2D gel electrophoresis^{39 (link)}. First dimension IEF done and strips focused on PROTEAN IEF unit (Bio-Rad) at 20 °C. Then strip were equilibrated and second dimension run on 12% SDS polyacrylamide gel at a constant voltage of 100 V for 3–4 h followed by staining with coomassie brilliant blue R250 for the visualization of proteins. Images of gel were analyzed with the help of Chemidoc [BIO-RAD] and proteins with different intensities were analyzed using PDQuest Advanced 2-D Gel Analysis software version 8.0.0 (Bio-Rad, Hercules, CA, USA).

The rehydration mix was brought to a final volume of 450 μL with rehydration buffer consisting of 5 M urea, 0.5% CHAPS, 0.5% Pharmalyte, and 12 μL/mL of DeStreak reagent (GE Healthcare). Each mixture was applied by rehydration loading to one IPG strip (ReadyStrip pH 3–10, nonlinear, 24 cm, Bio-Rad, Hercules, CA, USA) in the focusing tray of a Bio-Rad Protean IEF unit, sealed with silicone oil (Bio-Rad). The strips were rehydrated with the samples by “active rehydration” at 50 V and 20°C for 15 h and then focused for 3 h at 100 V, before the voltage was constantly increased to 8000 V within 17.5 hours, applying altogether 65 kVh with a maximum of 30 μA per strip.