Protein Separation and Characterization via IEF and SDS-PAGE

Isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were conducted as described previously [45 (link)]. The 18-cm immobibline dry strips (pH 4–7) (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were rehydrated within the BioRad Protean IEF Cell for 16 h at 20 °C with 300 μL rehydration buffer including 200 μg protein lysates respectively prepared from Sh-Gal-1(+120) and Sc-Gal-1(+120) T24 cells. Then, the proteins were concentrated at 20 °C at 50, 100, 200, 500, 1000, 5000, and 8000 V, respectively, with a total of 81,434 voltage-hours. After isoelectric focusing, the gel strips were equilibrated in the equilibration buffer (6 M urea, 30% (v/v) glycerol (Kanto Chemical, Portland, OR, USA), 2% (w/v) SDS (aMResco, Solon, OH, USA)) containing 2% (w/v) DTT for 15 min and then in the equilibration buffer containing 5% (w/v) iodoacetamide (aMResco, Solon, OH, USA) for a further 15 min. The equilibrated gel was loaded onto the top of a 12.5% (w/v) polyacrylamide gel and sealed with 0.5% (w/v) agarose (aMResco, Solon, OH, USA) and the proteins were separated at 420 V using BioRad Protean IIxi until bromophenol blue reached the bottom of the gel.