Amicon ultra 0.5 3 kda centrifugal filter units

The protein mixture obtained by TRIS extraction assisted with glass beads was purified, desalted, and concentrated by Amicon Ultra-0.5 3 kDa centrifugal filter units (Merck KGaA, Darmstadt, Germany). The concentrated solution was then subjected to protein precipitation using a precipitation kit (Ready-Prep™ 2-D Cleanup Kit, Bio-Rad, Warsaw, Poland) to obtain a pellet containing 200 µg of protein. The protein precipitate was dissolved in 300 µL of rehydration buffer containing thiourea (Bio-Rad, Warsaw, Poland), transferred to a rehydration plate, and covered with 17 cm immobilised pH gradient linear strips for isoelectric focusing (pH 3–10, Bio-Rad, Warsaw, Poland) flooded with mineral oil (pH 3–10, Bio-Rad, Warsaw, Poland) and left overnight [12 ].

For proteomic analysis, 7 individual urine sample from group I (DM I), 7 individual urine sample from group II (DM II) and 7 individual urine sample from healthy group (H) were collected.
Each urine samples were purified, desalted and concentrated by Amicon Ultra-0.5 3 kDa centrifugal filter units (Merck KGaA, Darmstadt, Germany). Protein concentrations were measured with a micro-volume spectrophotometer (MaestroNano, Maestrogen, Xinzhu, Taiwan), and the urine samples were then prepared and subjected to 2D electrophoresis. After graphical and statistical analysis protein of interest were cut from gel for further identification by mass spectrometry with the MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry) technique.