Chemidoc camera

Cellular pellet was lysed in lysis buffer (50 mM HEPES pH 7.5, 250 mM NaCl, 5 mM EDTA, 1% NP-40,1 mM DTT, 1 mM PMSF, 1X protease inhibitor cocktail (Roche)) and cells were incubated on ice for 30 min, vortexed every 5 min. Lysates were centrifuged at 18,000 × g for 1 h at 4 °C. The supernatant was transferred to a prechilled Eppendorf tube and stored at −80 °C. For protein electrophoresis, samples were heated in 1× SDS sample buffer for 5 min at 95 °C, loaded on a stain-free 4–15% SDS gel (Bio-Rad), and migrated at 130 V for 90 min in running buffer (1x Tris-Glycine, 0.1% SDS). The stain-free gel was visualized using a ChemiDoc camera (Bio-Rad). For transfer, a nitrocellulose membrane (VWR) was pre-equilibrated in dH₂O and transfer buffer (1x Tris-Glycine, 0.025% SDS, 10% methanol). The proteins were transferred for 2 h at 0.35 A at 4 °C. The membrane was blocked in 5% milk in 1× TBS-T at room temperature for 30 min and then incubated with the respective antibody (see antibodies below) in 5% milk in 1× TBS-T overnight at 4 °C. After extensive washes in TBS-T (3 × 10 min), the membrane was incubated for 1 h with the appropriate secondary HRP-antibody at room temperature on a shaker. After 3 more washes in TBS-T, the membrane was developed using ECL prime western blotting detection reagent (VWR) and visualized using a ChemiDoc camera (Biorad).