Calibrated imaging densitometer

The one-dimensional separation of total protein was performed using isoelectric focusing (IEF) according to a procedure mentioned in Bio-Rad manual book. 800 µg of total protein was taken for rehydration on 17-cm strips (Bio-Rad, Hercules, CA, USA) in a linear pH gradient (4–7). Isoelectric focussing (IEF) was conducted using the IEF Cell (Bio-Rad) with a procedure mentioned in Bio-Rad manual. The temperature of instrument during IEF was 20 °C and 50 mA electrical current was maintained per strip. After focussing, strips were equilibrated two times with equilibration buffer I and equilibrium buffer II, respectively, each for 15 min duration. Second dimension electrophoresis was performed with equilibrated strips in SDS-PAGE in a vertical slab of 12% acrylamide. PROTEAN Tetra Cell (Bio-Rad) instrument was used for these perpose. The protein gels were stained using the colloidal Coomassie Brilliant Blue R-350 and spots were visualised using a Calibrated Imaging Densitometer (Bio-Rad, GS–800). Three independent biological replicates were prepared for each of six sets of samples [WT and progeny transgenic plants prior to and post 24 and 48 h sheath blight infection].

Subsequent to staining, picture of the gel was captured using the instrument Calibrated Imaging Densitometer (Bio-Rad, GS–800) and the picture was thoroughly analysed with the help of PD Quest Software version 8.0 (Bio-Rad, USA). All the protein spots in the gel were compared to the respective spots in the reference gel, and normalisation of each spot density was performed against total densities of gel. The percentage volume of each spot was calculated considering the value of three biological replicates. Statistical analysis (t-test) was performed in order to evaluate the significant differences between WT and transgenic plants respectively. Only those protein spots showing reproducible fold change (>2.0-fold for up-regulation and ˂0.5-fold for down-regulation) and significant differences (P < 0.05) were considered as DEPs (differentially expressed proteins).