Protean 2 xl cell

Cells were lysed in 2-DE lysis buffer containing 7M urea, 2M thiourea, 4% CHAPS and 1% DTT (Sigma-Aldrich, St. Louis, MO, USA USA) supplemented with protease inhibitor cocktail (Roche, Switzerland). A total of 800 µg of proteins was solubilized in 2-DE rehydration buffer (7M urea, 2M thiourea, 4% CHAPS, 1% DTT and 0.2% Bio-Lyte ampholyte (Bio-Rad, Hercules, CA, USA), loaded onto 17 cm pH 4–7 IPG strips and subjected to isoelectric focusing on PROTEAN IEF cell (Bio-Rad). The IEF conditions were as follows: 50 V for 12 h, 200 V for 15 min, 500 V for 1 h, 500–10,000 for 3 h and 10,000 V for 5 h. In the second dimension, proteins were resolved by 12% SDS-polyacrylamide gels by PROTEAN II XL cell (Bio-Rad, Hercules, CA, USA). Gels were stained with Coomassie Blue G-250 (Sigma-Aldrich, St. Louis, MO, USA) overnight, and after washing in miliQ water, gel images were taken by ChemiDoc XRS+ Imager (Bio-Rad, Hercules, CA, USA). The 2-DE gel image analysis was carried out using Progenesis SameSpots 4.0 software (TotalLab, Newcastle upon Tyne, United Kingdom). The experiment was performed in four individual biological replicates for each cell line. ANOVA followed by post hoc Tukey’s test was carried out to identify statistically significant differences in protein abundance between the datasets obtained for the three cell lines differing in their BRAF mutational status.

A total of 900 μg of proteins extracted from each sample were used for 2-DE. First dimensional electrophoresis was performed using immobilized pH gradient (IPG) strips (pH 4–7, nonlinear, 17-cm ReadyStrip; Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. After isoelectric focusing, the IPG strips were equilibrated for 20 min in equilibration buffer (6-M urea, 20% w/v glycerol, 2% w/v SDS, and 50 mM Tris-HCl, pH 8.8) containing 1% w/v DTT, and then alkylated with 2.5% w/v iodoacetamide in equilibration buffer for 20 min. The strips were placed atop 12% w/v SDS-PAGE gels with no stacking gel. Twelve gels (three replicates for each treatment) ran simultaneously and electrophoresis was performed at 15°C and 3 W/gel for 1 h and then 15 W/gel using a PROTEAN II XL Cell (Bio-Rad). Gels were stained with Coomassie Brilliant Blue R-250, and then scanned using a GS-800 calibrated densitometer (Bio-Rad). The digitized protein spots on 2-D maps were quantitatively analyzed using PDQuest 2D analysis software (Bio-Rad) on the basis of their relative volumes. The optimized parameters were as follows: partial threshold, 4; saliency, 2.0; minimum area, 50. To verify the autodetected results, all spots were manually quantified by determining the ratio of the volume of each detected spot to the total volume of all spots on the gels.