Image master 2d platinum software version 6

Fifty micrograms of extracted protein was mixed with rehydration buffer (7 M urea, 2 M thiourea, 4 % CHAPS, 0.5 % (v/v) IPG buffer pH 3–10, 60 mM DTT and 40 mM Tris) and rehydrated into a 7-cm IPG strip (pH 3–10) for 10–15 h at room temperature. The first dimension separation or isoelectric focusing (IEF) was performed by the Ettan IPGphor III IEF System (GE Healthcare, Little Chalfont, UK) at 20 °C using a stepwise voltage increase to reach 9000 Vh. The focused IPG strip was equilibrated with an equilibration buffer (6 M urea, 130 mM DTT, 112 mM Tris–HCl pH 8.8, 4 % SDS, 30 % glycerol and 0.002 % bromophenol blue) for 15 min, followed by a second equilibration for 15 min in the same solution containing 135 mM iodoacetamide instead of DTT. The equilibrated strip was transferred to the top of a 12.5 % polyacrylamide gel and then second dimensional separation was performed using SE260 mini-Vertical Electrophoresis Unit at 150 V for approximately 2 h. Protein spots were visualized by silver blue CBG-250 staining [12 (link)]. The stained gel was scanned by Ettan DIGE Imager (GE Healthcare). Numbers of protein spot were detected using ImageMaster 2D-Platinum software version 6.0 (GE Healthcare).

Upon electrophoresis, Gels gels were stained with silver nitrate according to GE handbook (GE Healthcare, Uppsala, Sweden) with some modifications. Briefly, gels were fixed in 40% ethanol and 10% acetic acid for 60 min, and then sensitized with 30% ethanol, 0.2% sodium thiosulfate w/v, and 6.8% sodium acetate w/v for 30 min. Then gels were rinsed with distilled water three times, 5 min for each time, then incubated in silver nitrate (2.5 g/L) for 20 min. Incubated gels were rinsed with distilled water two times, and developed in a solution of sodium carbonate (25 g/L) with formaldehyde (37%, w/v) added (240 mL/L) for two times, first for 1 min, then stained for 4 min. Development was stopped with 1.46%w/v Ethylene Diamine Tetraacetic Acid for 10 min, then gels were rinsed with distilled water three times, 5 min for each time. Gels were stored in distilled water until they could be processed.Gels images were acquired using a PowerLook 2100XL color scanner (UMAX Technologies, CA, USA) and analyzed with Image master 2D Platinum Software Version 6.0 (GE Healthcare, Uppsala, Sweden).

Gels were fixed for about 8 h in solution (10% (v/v) acetic acid, 40% (v/v) ethanol, and 50% (v/v) water), washed 3 times in water, and then stained with Coomassie colloidal blue G-250 according to the GE handbook (GE Healthcare) with minor modifications. Gel images were acquired with the PowerLook 2100XL color scanner (UMAX Technologies, Atlanta, CA, USA) at a resolution of 16 bits and 300 dpi and were assayed by Image master 2D Platinum Software Version 6.0 (GE Healthcare). To compare the spot quantities between gels accurately, each spot volume was normalized as a percentage of the total volume of all of the spots in the gel. All automatic spot detections in each gel were manually inspected and edited as necessary to confirm the absence of mismatched and unmatched spots. Differentially expressed protein spots were changed abundance by at least ±1.2-fold, with an error probability of p < 0.05.