Decyder 2d software

To visualize the labeled and separated proteins after electrophoresis, the 2D-DIGE gels were scanned at a resolution of 100 µm with a Typhoon FLA 9500 (GE Healthcare). Subsequently, to visualize the total protein load, 2D-DIGE gels were fixed (1% citric acid, 30% ethanol) for 60 min and stained with colloidal Coomassie overnight (5% aluminum sulfate hydrate, 10% ethanol, 0.02% Coomassie G250, and 2% o-phosphoric acid). After destaining with deionized water until appropriate background reduction, gels were scanned with a visual scanner (CanoScan 9900F) using a resolution of 300 dpi.
For image analysis scan files of the 2D-DIGE gels were loaded into DeCyder 2D software (GE Healthcare, version 7.2). Spots were detected with an estimate of 5000 spots for the 2D-DIGE gel. Subsequently, a detection area excluding the region of strip application, molecular weight marker, and running front was determined. Spots with a volume below 50,000 were defined to be background. Stained crumbs originating from the dyes were eliminated by excluding spots with an area below 300. False positive spots, for example, produced by dye artifacts within the gel were removed manually. After editing the gels were normalized towards the Cy2 channel (internal standard).

The 2D-DIGE experiments were performed as per the protocol reported previously by Rapole and co-authors (31 (link)). Briefly, 60 µg of BMIF and serum proteins from controls and MM patients were labeled with 400 pmol of CyDyes Cy5, Cy3 respectively. Moreover, an internal control having 1:1 concentration of both samples was constituted and labeled with Cy2. The labeled samples were rehydrated on a 24 cm linear IPG strip (pH 4–7), and the strips were subjected to isoelectric focusing (IEF) through EttanIPGphor 3 instrument (GE Healthcare, USA). Following the first dimension IEF procedure, the second-dimension SDS-PAGE was performed and the gels were scanned through the Typhoon FLA 9500 biomolecular imager (GE Healthcare, USA). The images were analyzed using DeCyder 2D software; version 7.0 (GE Healthcare, USA) for both the differential in-gel analysis and biological variation analysis (BVA). The statistically significant protein spots passing the t-test (p-value ≤ 0.05) and present in all the gels were selected for MALDI-TOF/TOF identification.