Immobilon membranes

To determine the efficiency of protein expression in vitro, 4 μl of expression reaction were analyzed by Western blotting. The produced proteins were separated on SDS-polyacrylamide gels and then transferred onto Immobilon membranes (Thermo Fisher Scientific) using the mini-protein tetra cell system (Bio-Rad). After transfer, the membranes were blocked overnight at 4 °C with 5% (w/v) fat-free dry milk in PBS. The membranes were incubated for 1 to 3 h with the primary antibody (anti-GST or anti-Halo) at a 1:10,000 dilution in PBS containing 5% (w/v) fat-free milk and 0.05% (v/v) Tween 20. After washing (three times, 10 min each) with PBS containing 0.05% (v/v) Tween 20 to remove unbound primary antibody, the membranes were kept for 1 h at room temperature with the secondary antibody (mouse anti-goat IgG or anti-rabbit IgG fused to HRP at a 1:15,000 dilution in PBS containing 5% (w/v) fat-free milk with 0.05% (v/v) Tween 20. Excess of secondary antibody was removed by several washes (5–8 times, 15 min each) with PBS containing 0.05% (v/v) Tween 20. The presence of the fusion protein was detected using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and exposure to X-ray film (Research Products International Corp) according to the manufacturer’s recommendation. In some experiments, the detection of the bands was performed using ChemDoc MP (Bio-Rad).