The Western blot is widely used to detect and compare proteins. A single Western blot is often probed with multiple antibodies in a serial fashion. In order to avoid interfering signal from a previous experiment, the Western blot should be treated (i.e. stripped) to remove previous antibodies from the membrane.
For the last two years, I have been using Pierce’s Restore™ Western Blot Stripping Buffer for serial blotting of membranes containing samples of non small cell lung cancer cell line lysates. The use of the buffer is very straightforward. Blots should be kept at room temperature before adding the stripping buffer. The blots to be stripped are incubated with the Restore™ Western Blot Stripping Buffer for 5-10 minutes; the volume should be sufficient to completely wet the membrane. Generally, approximately 20 ml of this buffer is required for an 8 x 10 cm blot. The washing procedure is very simple. After draining off the excess Restore™ buffer, we washed the stripped membrane with water for 5 minutes, at least 2 times in order to remove the Restore™ buffer. The membrane should be thoroughly washed before blocking with 5% milk. The whole process (stripping and washing) takes about 20 minutes. When we did not want to use the stripped membrane right away, we stored the stripped membrane in TBS at 4ºC for future use.
The use of this buffer is much better than traditional stripping methods as the Restore™ buffer generally provides a robust, but gentle method for stripping antibodies unlike the traditional stripping method. The traditional method I used before was the standard beta-mercaptoethanol/SDS protocol. With this traditional method, sometimes we ended up having an incomplete stripping of antibodies. By using this Pierce’s Restore™ Western Blot Stripping Buffer, we were able to overcome this problem. The Buffer works the best when the blot has been previously used for detection of protein using SuperSignal West Chemiluminescent substrates.
This product is not very expensive; however, it is not cheap either. (Please note that this buffer is not good for the purpose of dissociating a biotinylated target protein and avidin conjugate probes.)
In summary, I highly recommend Pierce’s Restore™ Western Blot Stripping Buffer for the purpose of serial probing with multiple antibodies using the same nitrocellulose or PVDF membrane. It is reliable if properly used and the process is very time efficient.
Saswati Hazra, PhD
Postdoctoral Fellow
University of California, Los Angeles (UCLA)
Department of Medicine