Stripping and Reprobing a Western Blot Membrane

Last updated: 1/12/12 By: Tim Starr

Overview

Follow this protocol to strip off antibodies from a membrane in order to re-stain the membrane with a different antibody. Generally use mild stripping, unless it doesn't work. PVDF membranes are more durable and will withstand more stripping and reprobing. Can check efficiency of stripping by incubating with chemiluminescent reagent and image the blot. This protocol comes from the Abcam protocol

Protocol 1: Mild stripping

• Place membrane in container and rock at room temperature for each step with enough liquid to cover membrane
• Add stripping buffer and rock for 5 minutes, discard buffer
• Add fresh stripping buffer for 5 minutes, discard buffer
• PBS 10 min
• PBS 10 min
• TBS-T 5 min
• TBST 5 min
• Membrane is now ready for blocking and protein detection

Mild stripping buffer (100 ml)

Protocol 2: Harsh stripping

• When making the buffer, add Beta-mercaptoethanol in fume hood and keep containers closed
• Warm buffer to 50º C
• Cover membrane either in container or seal-a-meal bag
• Incubate 45 minutes at 50ºC with some agitation
• Rinse under running tap water for 1 to 2 hrs
• Wash with TBS-T for 10 minutes
• Membrane is now ready for blocking and protein detection

Harsh stripping buffer (100 ml)