2.4. Isoelectric focusing

The pI value of the succinylated LYS analogues were determined by isoelectric focusing (IEF) using Novex™ pH 3–10 IEF Protein Gels, 1.0 mm, 10 wells, and the XCell SureLock™ Mini Gel Tank. The anode and cathode buffers were purchased from Thermo Fischer Scientific as a part of the Novex™ pH 3–10 IEF Buffer Kit and cooled to 4 °C before use. The samples were mixed with sample buffer (1:1, v/v) at a concentration leading to minimum 2 µg protein on the gel. The used marker was IEF Marker 3–10, SERVA Liquid Mix (ThermoFisher Scientific). The gel was run at 100 V for 1 h, followed by 200 V for 1 h and 500 V for 30 min. The gel was fixed with a 12% (v/v) trichloroacetic acid solution (TCA 20%, Carl Roth, Karlsruhe, Germany) for 30 min followed by staining using Bio-Safe™ Coomassie brilliant blue G-250 (GE Healthcare) solution for 1 h. The gel was destained in water for 3 × 5 min.