|"Protein expression profiling was performed using 2D-DIGE and the Auto2D. In brief, 1 Âµg of each HuO9 protein lysate and the internal standard sample were labeled with Cy5 and Cy3 fluorescent dye (CyDy DIGE Fluor saturation dye, GE Biosciences, Uppsala,Sweden), respectively. After stopping the labeling reaction, the labeled samples were mixed, and made up to 10 Âµl with lysis buffer containing 0.5% ampholyte (GE Biosciences) and 20 mM DTT. The sample was then applied to the Auto2D in accordance with the manufacturerâs instructions. The running conditions were programmed as follows: sample application by the rehydration method, 35 min; first-dimension separation, 40 min; equilibration, 10 min; and second-dimension separation, 35 min. The equilibration buffer contained 475 mM Tris- HCl (pH6.6), 3.8% SDS, 47.5 mM DTT, 11.875% glycerol, 0.00475% BPB. The first dimension separation was done at 200V constantlyfor 5 min, gradually increased to 1000V for 5 min, constantly 1000Vfor 5 min, and gradually increased to 7000V for 10 min, and constantly 7000V 15 min.
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Citation: Tani Y, Tajima T, Kawai A, Unuma Y , Kinoshita H, et al. (2014) Evaluation of a Novel Automated Machine, the Auto2D, for Two-Dimensional Gel Electrophoresis. J Proteomics Bioinform 7: 108-111."