Urinary protein separation by one-dimensional SDS gel and reverse-phase HPLC. (a) 150 μg urinary protein (25 μg/lane) from single sample and pooled sample were applied on a 4-12% Bis-Tris gel. Gel was stained by colloidal Coomassie and cut into 14 pieces (in-gel 1 set) or 10 pieces (in-gel 2 set) for single urine sample, and cut into 10 pieces for pooled urine sample. (b) 250 μg of urinary protein was applied to Vivapure Anti-HSA Kit to deplete serum albumin. The albumin-depleted protein mixture was dissolved in 6 mol/l urea and 1.0% acetic acid solution, and separated on mRP-C18 High-Recovery protein column at 80°C using linear multi-segment gradient, as described in the Materials and Methods section. HPLC, high-performance liquid chromatography; SDS, sodium dodecyl sulfate.