2.6.3. SDS-PAGE and Western blot
SDS-PAGE was performed using 10% polyacrylamide handcast gels in the Mini-Protean Tetra Cell system (BioRad, USA) operating at 150V and 3.0 A for 90 minutes. A 4-fold concentrated sample buffer (0.25 M Tris-HCl, 40 g/L SDS, 20% v/v glycerol, 0.5 g/L bromophenol blue) was added (1:4) to the samples, which were then heated at 99 °C for five minutes before applying 40 L of each sample to the gel. The tank was filled with running buffer (3.04 g/L Trizma®-base, 14.6 g/L glycine, 1 g/L SDS, pH 8.4). The gel was silver stained and photographed.
For the Western blot, the gel and a nitrocellulose membrane were wetted with transfer buffer (5.82 g/L Trizma-base, 2.93 g/L glycine, 20% v/v methanol) and proteins were transferred to the membrane using a Trans-Blot® SD Semi-Dry Transfer Cell (BioRad, USA) set at 15 V and 3.0 A for 90 minutes. After the transfer, the membrane was blocked using a 5% (m/v) skimmed milk solution followed by incubation with 4G2 antibody (MAB10216, Merck, USA) diluted 1:8,000 and anti-mouse IgG HRP-conjugate (A16011, Invitrogen, USA) diluted 1:10,000. Then the membrane was incubated with ECL Prime detection reagent (GE Healthcare, USA) and image was obtained using a FluorChem E system (ProteinSimple, USA).