How to strip and reprobe your western blot

Stripping and reprobing a western blot save time and conserves samples while allowing for optimization or detection of a different target protein. With chemiluminescent and fluorescent detection, the signal-producing chemicals can be washed away, and the antibody stripped from the membrane so that the blot can be reprobed for another target protein. If you’re going to be performing multiple strips and reprobing for different antigens, probe for the low-abundance proteins first. Various stripping buffer formulations are commercially available or can be made in the lab. These buffers generally include a detergent and reducing agent. Some formulations require heat and/or low pH to effectively strip off antibodies. To strip a blot of all antibodies and or chemiluminescent reagents, immerse the blot in stripping buffer and either incubate with agitation or heat in an oven. If you are using Thermo Scientific™ Restore™ buffer, incubate the blot for 5 to 10 minutes at 37 degrees Celsius. If using Restore for Fluorescent blots, incubate 10–15 minutes at room temperature. After the specified incubation period, wash the blot thoroughly to ensure that all of the stripping buffer is removed. When using stripping buffers with beta-mercaptoethanol as the reducing agent, you will know that your blot has been sufficiently washed when you can no longer smell the pungent aroma of the reagent. Once stripped, the blot will need to be re-blocked before the second immunoprobing experiment can be performed. Learn more information about western blot stripping buffers at thermofisher.com/western buffers