Imagemaster 2d elite software

2-DE samples were prepared according to the previously described methods.^{25 (link), 26 , 27 (link), 28 (link), 29 (link)} After delipidation and desalting, the protein concentration of the samples was measured via a modified Bradford method using BSA as a standard.^{30 (link)} Immobilized DryStrips (24 cm, pH 3-10) utilized for isoelectric focusing (IEF) were rehydrated with 40 μg of protein in 450 μl of solubilization solution containing 8 M of urea, 2% of CHAPS, 1% of immobilized pH gradient (IPG) buffer (pH 3-10), 13 mM of dithiothreitol (DTT) and a trace of bromophenol blue for 5 h without current and for another 7 h at 80 V. IEF was conducted using the IPGphor IEF system (GE Healthcare, Uppsala, Sweden) for 120 000 Vhr. The second dimension was run on 12.5% of SDS-PAGE with an Ettan DALT II system (GE Healthcare). Proteins were visualized via silver staining. All experiments were conducted in triplicate. Computer analyses of the 2-DE images were conducted using an ImageMaster 2D Elite Software (GE Healthcare). The expression levels of the spots were determined in accordance with the relative spot volume of each protein, as compared with the normalized volumes of proteins.^{31 (link)}

To this set of experiments, soluble extracts (10 µg) from Dm28c were separated by 12% SDS-PAGE co-polymerized with 0.1% gelatin (Heussen and Dowdle, 1980 (link)). Samples were diluted (v/v) in SDS-PAGE sample buffer 4× without reducing agent and loaded onto gels. Electrophoresis was carried out under constant current (25 mA) at 4 °C. The gels were then soaked in 2.5% Triton X-100 under agitation for 1 h at room temperature for SDS removal and enzyme renaturation. The effect of pH on the proteolytic activity was determined by incubating the gels for 24 h at 37 °C in 50 mm phosphate buffer (pH 5.5 and 7.4) and 100 mm glycine-NaOH buffer pH 10.0 containing or not 2 mm DTT. Then, the gels were stained with 0.2% Coomassie Brilliant Blue R-250 in methanol:acetic acid:water (40:10:50) and destained in the same solution without the dye. The gels were scanned with the Image Scanner III (GE HealthCare) and analysed by the Image Master 2D Elite software (GE HealthCare). To determine the peptidase class, the lysates were pre-incubated for 30 min with the following inhibitors for 30 min at room temperature: pepstatin A (1 µm) for aspartic-peptidases, phenanthroline (10 mm) for metallo-peptidases, PMSF (1 mm) for serine-peptidases and E-64 (10 µm) for cysteine-peptidases. These inhibitors were also included in the pH 10.0 buffer in which the zymograms were incubated overnight.