Western blot analysis of Atxn7 using anti-Atxn7 antibody (DZ41648).
Electrophoresis was performed on a 4–20% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 µg of protein sample mixed with 4× Laemmli Sample Buffer and 2-mercaptoethanol and heated at 95 °C for 5 min prior to loading.
Lane 1: mouse 3T3 cell (WT),
Lane 2: mouse 3T3 cell (WT + Dox),
Lane 3: mouse 3T3 cell (Scramble shRNA),
Lane 4: mouse 3T3 cell (Scramble shRNA + Dox),
Lane 5: mouse 3T3 cell (Atxn7 shRNA),
Lane 6: mouse 3T3 cell (Atxn7 shRNA + Dox),
Lane 7: mouse 3T3 cell (WT),
Lane 8: mouse 3T3 cell (WT + Dox),
Lane 9: mouse 3T3 cell (Scramble shRNA),
Lane 10: mouse 3T3 cell (Scramble shRNA + Dox),
Lane 11: mouse 3T3 cell (Atxn7 shRNA),
Lane 12: mouse 3T3 cell (Atxn7 shRNA + Dox).
After electrophoresis, proteins were transferred to 0.45 μm PVDF membrane at 150 mA for 50-90 minutes. Blocked the membrane in 5% non-fat dry milk in TBST (1X TBS with 0.05% Tween-20) for 30 min at RT. The membrane was incubated with rabbit anti-Atxn7 antigen affinity purified polyclonal antibody (DZ41648) at 1:1000 overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:4000 for 1 hour at RT. The signal is developed using SuperSignal West Pico PLUS and imaged on a ChemiDoc MP system. A specific band was detected for Atxn7 at approximately 100-130 kDa.