Tran blot sd semi dry transfer cell

We incubated 0.05 μM E1 (Ube1), 1 μM E2 (UbcH5b), 2 μM WT or mutant Zn-RING-Zn domain and 20 μM WT or mutant Ubiquitin at 37°C for 3 h in a reaction buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 10 mM MgCl_2, 5 mM ATP and 1 mM DTT. For substrate ubiquitination GST-Numb was added to the above reaction mixture at 2 μM concentration. The reaction was quenched using SDS loading buffer and was resolved using 12.5% SDS-PAGE. The protein bands were transferred to PVDF membranes using a Tran-Blot SD Semi-Dry Transfer Cell (Bio-Rad). The membrane was then probed with anti-ubiquitin or anti-LNX2 or anti-Numb antibody in 1:2000 dilutions followed by washing and then incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibodies in a 1:10,000 dilution. The bands were visualized using ECL western blotting substrate (Pierce) as instructed by the manufacturer.

SDS–PAGE gels were transferred to PVDF membranes (Sigma‐Aldrich) using a Tran‐ Blot SD Semi Dry Transfer Cell (Bio‐Rad). The blocking of membranes was performed with 5% BSA in phosphate buffered saline (PBS) containing 0.05% Tween 20 (PBS‐T) for 60 min. Membranes were then incubated with mouse monoclonal anti‐RAC1 or anti‐ubiquitin antibody (Santa Cruz Biotechnology, SantaCruz, CA) at 1:2000 dilutions at 4 °C overnight. Subsequently, membranes were subjected to three washes with PBS‐T and incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (Jackson ImmunoResearch) in a 1:10 000 dilutions for 60 min at room temperature (23 °C). Membranes were further washed three times with PBS‐T and added with Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific). Subsequent visualization was conducted with GeneSys image acquisition software with Syngene Pxi system.