hrCN PAGE Protein Separation Protocol

The hrCN PAGE protocol was adapted from Lemaire et al. (2018) [24 (link)] Glycerol was added to the sample at a final amount of 20% v/v. Ponceau S at a final concentration of 0.001% w/v served as a marker to follow the migration. The buffer composition for the electrophoresis cathode was the following: 50 mM Tricine, 15 mM Bis-Tris/HCl, pH 7, 0.05% w/v sodium deoxycholate and 0.01% w/v dodecyl maltoside, while the anode buffer contained 50 mM Bis-Tris/HCl buffer pH 7. A 5 to 15% linear polyacrylamide gradient gel was used and electrophoresis was run with a constant 40 mA current (PowerPac^TM Basic Power Supply, Bio-Rad). After electrophoresis, protein bands were visualised with Ready Blue^TM Protein Gel stain (Sigma Aldrich, Hamburg, Germany). The native protein ladder used is NativeMark^TM Unstained Protein Standard (ThermoFischer Scientific, Dreieich, Germany).
Determination of the oligomeric state by gel filtration was performed on a Superose6 Increase 10/300 GL (GE Healthcare, Munich, Germany) in 25 mM Tris/HCl pH 7.4, 2 mM DTT, 10% glycerol at a 0.4 mL/min flow rate. High Molecular Weight range Gel Filtration Calibration Kit (GE Healthcare, Munich, Germany) was used as the protein standard.