Decyder

Two-dimensional DIGE was performed at Applied Biomics (Hayward, CA, USA) following typical methods [19 ,20 (link)]. Briefly, cell lysates, were denatured by equal volume addition of lysis buffer containing 7M urea, 2M thio urea, 4% 3-((3-cholamidopropyl)dimethyl ammonio)-1-propanesulfonate(CHAPS) followed by addition of 30 mM Tris-HCl, pH 8.8, at a 5:1 volume ratio lysis buffer: plasma. Lysate samples were normalized using total protein as determined by Lowry protein estimation method. Next, samples were labeled with CyDye DIGE fluors developed for fluorescence 2D-DIGE technology (Cy3 and Cy5, GE Healthcare, CT, USA) and incubated in dark on ice, 30 min. The labeled samples were then subjected to isoelectric focusing (IEF) on a 13-cm precast non-linear immobilized pH gradient strip (pH 3-10, Amersham Biosciences, Buckinghamshire, UK) using an Amersham Pharmacia IPGPHOR unit with a power supply (EPS3501XL) in gradient mode. Next, the samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) in the second dimension based on size. The gels were scanned using Typhoon Trioscanner (Amersham Biosciences) and fluorescent dye signals corresponding to individual samples converted to black and white images for analysis using Image Quant and DeCyder software (Amersham Biosciences).