Typhoon fla 9500 scanner

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).

Four groups of proteins (prepubertal GnRH-A, prepubertal hCG, mature GnRH-A, and mature hCG) of preovulatory follicle walls (granulosa and theca layers) were resolved using 2D-DIGE. The obtained gels were scanned with a Typhoon 9500 FLA scanner (GE Healthcare) using the parameters suggested by the manufacturer’s instructions. The scanned images were analyzed with DeCyder Differential Analysis software version 5.02 (GE Healthcare) to identify differences in fluorescence intensities of the spots. During spot detection, the estimated number of spots was set at 10,000 and volume < 30,000. Protein spots with a P < 0.05 by one-way analysis of variance (ANOVA), which indicated an increase or decrease in relative intensity (in-gel ratios greater than 1.15), were considered differentially abundant proteins. Only spots that were successfully matched on > 80% of the gel images were considered for further analysis. To properly select and identify the spots, gels were stained using Coomassie Brilliant Blue G250 after 2D-DIGE.

After 2D-DIGE electrophoresis, the CyDye-labelled gels were scanned with a Typhoon 9500 FLA scanner (GE Healthcare) using the parameters recommended by the manufacturer. The SameSpots software (Totallab, Newcastle, UK) was used to match and analyze protein spots. Gels were aligned automatically and the alignment was refined manually. Differential in-gel analysis was used to calculate protein abundance alterations between samples on the same gel. The resulting spot maps for each biological replicate were then analyzed through biological variation analysis to provide statistical data on the differential protein expression. Spots that exhibited a change of the cumulated normalized abundance from all replicates of at least 2.0 and a p value < 0.05 were considered as differentially regulated.

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).

After electrophoresis, the gels were scanned with a Typhoon 9500 FLA scanner (GE Healthcare) at excitation/emission wavelengths of 532/576 nm (S-Dye200) and 633/664 nm (S-Dye300), respectively. SameSpots software (TotalLab, Newcastle upon Tyne, England, UK) was used to detect, normalise and quantify the spot patterns of all SDS-PAGE and 2D-PAGE gels. A Student’s t-test analysis embedded in the software was used to detect differences in the degree of cysteine oxidation of the proteins manifested in the differences in spot fluorescence in relation to the internal standard. Proteins for which the intensity changed (p < 0.05, fold change >2) were selected for further mass spectrometry identification. To properly select and identify the spots, additional preparative gels were run using 200 μg of pooled proteins. Proteins were localised and identified as previously described [67 (link)].

Protein samples were loaded onto 24-cm Immobiline DryStrips, with a 3–10 nonlinear gradient pH range (GE Healthcare, Uppsala, Sweden), and rehydrated for 12 h. In the first dimension of electrophoresis, the proteins were separated according to their isoelectric point using an Ettan IPGphor apparatus (GE Healthcare, Uppsala, Sweden) at 20 °C with current limited to 50 µA per strip and the following voltage program: 500 V over 2 h, a linear gradient to 1000 V over 1 h and a linear gradient to 10,000 V over 3 h, then at 10,000 V constant for 4 h. Subsequently, the strips were equilibrated in SDS equilibration buffer (6 M urea, 75 mM Tris-HCl, pH 8.8, 29.3% glycerol, 2% sodium dodecyl sulfate, 0.002% bromophenol blue) containing 65 mM DDT for 15 min, and then in SDS equilibration buffer containing 135 mM iodoacetamide for 15 min. The equilibrated strips were then transferred to precast DIGE gels Ettan DALT Gel 12.5 (25.5 × 19.6 cm, 1 mm thickness, GE Healthcare, Uppsala, Sweden) and sealed with 0.5% agarose. A second dimension of electrophoresis was then performed at 1.5 W/gel in an Ettan Dalt-Six apparatus (GE Healthcare, Uppsala, Sweden) for 16 h. The gels were scanned using a Typhoon 9500 FLA scanner (GE Healthcare, Uppsala, Sweden) and the obtained gel images were subjected to a statistical analysis.