Do you have a recommended protocol for stripping/re-probing blots?

The western blot stripping/re-probing protocol for our Western Blot Stripping Buffer (5X) #91925 is below. However, it is important to note that we typically advise against stripping/re-probing because stripping can be incomplete and/or lead to inconsistent results through the loss of target proteins from the membrane. If possible, it is much better to run fresh immunoblots with each individual antibody on duplicated sets of samples. 

A.    Solutions and Reagents 
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

Supplied Reagents:
1. Western Blot Stripping Buffer (5X) #91925: To prepare 20mL of 1X Stripping Buffer, combine 4mL 5X Stripping buffer with 140uL 2-mercaptoethanol (0.1M final concentration) and 15.86mL RODI water.

Additional Reagents (Not Supplied):
1. 10X Tris Buffered Saline with Tween® 20 (TBST) #9997: To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
2. 2-mercaptoethanol

B. Stripping Membranes
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different-sized membranes, adjust volumes accordingly.
NOTE: This protocol begins after membrane exposure to film or digital imager. The best results are obtained if the membrane is not allowed to dry. Membranes can be stored in 1X TBST at 4°C.
NOTE: Quantitative comparison of targets before and after stripping is not recommended due to the removal of small amounts of membrane-bound protein with each round of stripping.
1. Wash three times for 5 min each with 15 ml of 1X TBST at room temperature.
2. Incubate membrane in 20 ml of Stripping Buffer for 45 minutes to
1 hr at 50°C with slight agitation, if available. Depending on antibody signal strength, optimization of incubation time and temperature may be required. The incubation time can be adjusted up to 2 hours while the incubation temperature can be adjusted to 70°C if needed.
3. Wash five times for 5 min each with 15 ml of TBST at room temperature.
4. (Optional) To check the efficiency of the stripping and confirm that the original signal has been removed, incubate the membrane with the appropriate secondary antibody and proceed with protein detection using recommended detection reagents. If stripping is complete, continue by washing the membrane five times for 5 min each with 15 ml of TBST at room temperature. If stripping is insufficient, repeat steps 2 through 4.
5. The membrane is ready for reuse. Proceed with the blocking step before adding the primary antibody.
 

Last updated: September 12, 2024