Western Blot Reprobing Protocol
A. Solutions and Reagents
NOTE: Prepare solutions with Milli-Q or equivalently purified water.
- Wash Buffer: 1X Tris buffered saline, 0.1% Tween-20 (TBS/T)
- Stripping Buffer: To prepare 100 ml, mix 0.76 g Tris base, 2 g SDS and 700 μl β-mercaptoethanol. Bring to 100 ml with deionized H2O. Adjust pH to 6.8 with HCl.
- After film exposure, wash membrane four times for 5 minutes each in TBS/T. Best results are obtained if the membrane is not allowed to dry.
- Incubate membrane for 30 minutes at 50°C in stripping buffer (with slight agitation).
- Wash membrane six times for 5 minutes each in TBS/T.
- (Optional) To assure that the original signal is removed, wash membrane twice for 5 minutes each with 10 ml of TBS/T. Incubate membrane with LumiGLO® with gentle agitation for 1 minute at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.
- Wash membrane again four times for 5 minutes each in TBS/T.
- The membrane is now ready to reuse. Start detection at the “Membrane Blocking and Antibody Incubations” step in the Western Immunoblotting Protocol.
posted June 2005