2 O on top of the gel solution. When the stacking gel has polymerized, set up the running unit. Run the gel at 120 V for 1 hour. After running the gel, put the gel in transfer buffer for about 20 min with gentle shaking. Soak the nitrocellulose membrane in the transfer buffer for about 10 min.
All components are pre-wet in transfer buffer.
Put cold transfer buffer and the ice cooler into the chamber. We are going to do the overnight transfer. Stain the membrane in Ponceau S solution for 10 min and rinse with water. Wash the membrane with TBS or Blotto for three times with gentle shaking for 5-10 minutes each time. Wash with 1X TBS for three times with gentle shaking for 5-10 min.