Versa doc

D. regia pollen proteins from 7 cm 2D gel were transferred onto PVDF membrane by semi-dry transfer method as described earlier in specific IgE immunoblot. After transfer, the membrane was blocked with 3% BSA and immunoblotted with pooled sera from 17 atopic patients. Another blot was done using pooled sera from healthy subjects as a negative control. Images of the blots were acquired in Bio-Rad Versa Doc (Bio-Rad Laboratories) system, and molecular weights of reactive spots were determined by Quantity One software (version 4.6.3, Bio-Rad Laboratories). Sero reactive spots with high signal (intensity value greater than 2) were considered for further proteomic analysis.

D. regia pollen protein extract of around 400 μg was processed using Focus Perfect 2D Clean up Kit (G Biosciences, St. Louis, MO, USA) following manufacturer's protocol and then reconstituted in 125 μL rehydration buffer (IEF) containing 1% immobilized pH gradient (IPG) buffer for pH 4–7 (v/v) (GE-Healthcare), 25 mM DTT and traces of Bromophenol blue. The sample was applied to 7 cm Immobiline Dry Strip (IPG strip, pH 4–7 L) (GE-Healthcare) in a re-swelling tray and left overnight at room temperature. Isoelectric focusing was performed next day using Ettan IPG phor-3 isoelectric focusing system (GE-Healthcare) as per manufacturer's protocol. Briefly, the strip was focused at 0–200 V for 1 min, 200–3500 V for 1.30 h and 3500 V for 1.15 h, with a total of 8 kV accumulated. Next day the strip was equilibrated with equilibration buffer – I (6 M Urea, 75 mM Tris-Cl pH 8.8, 30% glycerol, 2% SDS and 1% w/v DTT) for 15 min followed by equilibration buffer-II (same as equilibration buffer-I with 2.5% w/v iodoacetamide instead of DTT). Finally, the strip was run in 12% SDS-PAGE with mini vertical Electrophoresis System (GE-Healthcare). The gel was then stained with Coomassie Brilliant Blue- G250 and photographed in a Bio-Rad Versa Doc (Bio-Rad Laboratories) system.