Decyder differential in gel analysis version 5

After electrophoresis, the stained 2D gels were scanned on a Typhoon 9500 FLA scanner (GE Healthcare) using the parameters recommended by the manufacturer for 2D-DIGE experiments. Image analysis was performed using the DeCyder Differential In-Gel Analysis version 5.02 software (GE Healthcare) in order to identify fluorescent areas. The DeCyder biological variation analysis module was applied to detect protein spots and concurrently match all twelve protein spot maps from six gels using several parameters: 1.) estimated number of spots at 10,000 and 2.) minimum spot size at 3,000. Only protein spots with a P<0.05 by t-test analysis that showed at least a 1.2-fold increase or decrease in their relative intensities in any comparison between all groups were significantly different. To properly pick and identify the selected spots, DIGE gels were stained using Coomassie Brilliant Blue G-250 (Bio-Rad, Hercules, CA).

After electrophoresis, the gels were scanned with a Typhoon 9500 FLA scanner (GE Healthcare) using the parameters suggested by the manufacturer for 2D-DIGE experiments. The scanned images were analysed with DeCyder Differential In-Gel Analysis version 5.02 software (GE Healthcare) to identify the fluorescence intensities of the spots. The DeCyder biological variation analysis module was used to detect protein spots, simultaneously matching all 24 protein spot maps from 12 gels using the following parameters: the estimated number of spots was set to 10,000 and the minimum spot size was set to 3,000. Protein spots with a p-value <0.05 by one-way ANOVA analysis, which showed an increase or decrease in relative intensity, were considered to be differentially abundant proteins. Only spots that were successfully matched on >80% of the gel images were considered. To properly select and identify the spots, gels were stained using CBB-G250 after 2D-DIGE, followed by spot excision and identification using matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS).