Western Blot Reprobing Protocol

A. Solutions and Reagents

NOTE: Prepare solutions with Milli-Q or equivalently purified water.

1. Wash Buffer: 1X Tris buffered saline, 0.1% Tween-20 (TBS/T)
2. 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.

B. Protocol

1. 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.
2. Incubate membrane for 30 minutes at 50°C in stripping buffer (with slight agitation).
3. Wash membrane six times for 5 minutes each in TBS/T.
4. (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.
5. Wash membrane again four times for 5 minutes each in TBS/T.
6. The membrane is now ready to reuse. Start detection at the “Membrane Blocking and Antibody Incubations” step in the Western Immunoblotting Protocol.