Pdquest 2 de analysis software

The 2-D gels stained by Coomassie brilliant blue (CBB) R250 [containing 50% (v/v) of methanol, 15% (v/v) of acetic acid and 0.1% (w/v) of CBB-R250] were scanned using an EPSON PERFECTION V700 PHOTO scanner (Epson), and protein spots of 2-D gel with different abundance changes were analyzed using PDQuest 2-DE Analysis Software (BIO-RAD, USA). The protein spots (fold change ≥2) were used for trypsin digestion and MALDI-TOF-MS analysis with AXIMA-CFR plus (Shimadzu Biotech, Kyoto, Japan) as reported by Li et al. (2012 (link)) and Shi et al. (2013a (link)). MASCOT software (Mascot Wizard 1.2.0, Matrix Science Ltd., http://www.matrixscience.com) was used to analyze the MS data. Since bermudagrass is an un-sequenced species, the homologous proteins were blasted against sequenced plant species. In the searching process against NCBInr and Swiss-Port protein sequence databases, peptide masses were assumed to be monoisotopic, and 100 ppm was used as mass accuracy, and one missing cleavage site was the maximum, and modifications were also considered. The minimum score of 43 and the minimum sequence coverage of 6% in MOWSE analysis were used to keep the confidence of the identification results.

Briefly, total protein extractions of H1299 cells was directly lysed in rehydration sample buffer (8 M Urea, 50 mM dithiothreitol (DTT), 4% 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate (CHAPS), 0.2% carrier ampholytes) as provided by Bio-Rad Laboratories (Hercules, CA) and were vortexed vigorously for 1 h at room temperature (RT). Insoluble substances were removed by centrifuge at 16,000 × g for 30 min at 4°C. Supernatant was collected and protein concentration was measured by the Bradford assay (Bio-Rad, Hercules, CA). A total of 200 μg protein was applied on a pH 3–10, 11-cm isoelectric focusing (IEF) strip (Bio-Rad, Hercules, CA). IEF was performed at a current of 50 mA per gel, 300 V for 30 min, followed by 3,500 V for 2.5 h, and additional 8,000 V for 5 h. Strips were immediately stored at −80°C for the second dimensional gel electrophoresis (2-DE) analysis. For the second dimensional electrophoresis, 10% SDS-polyacrylamide gels (SDS-PAGE) were used. Proteins were transferred onto nitrocellulose membrane (Osmonics Inc., MA) for subsequent Western blotting analysis or stained with 0.1% Coomassie blue R-250 prepared in 40% methanol/10% acetic acid. The spots were visualized using PDQuest 2-DE analysis software as described in the manufacturer's manual (Bio-Rad, Hercules, CA).

The 2-DE gels were scanned at 600 DPI resolutions with an EPSON PERFECTIONTM V700 PHOTO scanner (Epson (china) Co., Ltd.). The gel images were analyzed with PDQuest 2-DE Analysis Software (Version 8.0, Bio-Rad, USA). Individual spot volume was normalized by total spot volumes per gel to eliminate experimental variations among different 2-DE gels. The average volume of each spot in different experimental treatment was calculated from three biological replicates. The protein spots expression with more than 1.5-fold change (P<0.05) among different experimental treatment were regarded as receivable.