Commercial stripping buffers, such as G-Bioscience’s Western ReProbe™ and Western ReProbe™ PLUS, are specifically formulated to dissociate and remove all primary and secondary antibodies from the membrane-immobilized proteins without destroying the antigenic binding sites or removing the protein. This allows for several reprobings on the same membrane which will save you time, money, and precious protein samples. Western blotting can be time consuming (protein electrophoresis (1-2 hrs) + protein transfer (0.5 hrs to overnight)), so you want to make the most of your precious membranes and proteins by stripping and reprobing your nitrocellulose or PVDF membrane. There are many advantages to stripping your membrane such as: conserving limited/expensive protein samples, using different antibodies to analyze the same sample and confirm results, compare phosphorylated and total protein on the same blot, recoup antibody for later use, run less SDS-page gels and transfers, and to remove/adjust the concentration of your antibody.
There are many advantages to using G-Bioscience’s Western ReProbe™ and Western ReProbe™ PLUS commercial stripping buffers over traditional stripping methods. Western ReProbe™ (mild stripping agent) and Western ReProbe™ PLUS (strong stripping agent for stubborn, high affinity antibodies) have straightforward protocols that gently break the antigen-antibody binding affinity without using denaturants (ß-mercaptoethanol and Dithiothreitol (DTT)), sodium dodecyl sulfate (SDS), or boiling. Therefore, your target protein is conserved for multiple reprobings. Researchers have used Western ReProbe™ and Western ReProbe™ PLUS to strip and reprobe immunoblots as many as three consecutive times. Traditional stripping methods involve incubating your membrane with a low pH glycine solution (30-120 minutes at room temperature or 37°C) for mild stripping and/or using a combination of heat and detergent, and denaturants (30 minutes at 70°C) for harsh stripping. These traditional methods will inevitably cause some denaturation and loss of target protein. Furthermore, recovery of your stripped antibody is not possible when using heat, detergents and denaturants to strip your membrane. Western ReProbe™ and Western ReProbe™ PLUS also saves you valuable time by only requiring a 30 minute incubation period at room temperature to efficiently dissociate probe interactions, including chemiluminescent and radio-isotopic signals from blots, on nitrocellulose or PVDF membranes. The membrane bound proteins are retained on the membrane while the antibodies are washed away. The membrane bound proteins are now free to accept new antibodies.
- Western ReProbe™ is supplied as 5X solution, 100ml is sufficient reagent for 25-30 standard (7.5cm x 8.5cm) western blots.
- Each 100ml of Western ReProbe™ PLUS is sufficient for 25‐30 standard size (7.5cm x 8.5cm) western blots.
Some important considerations before you start stripping and reprobing:
- PVDF membranes are recommended over nitrocellulose membranes because they are more durable and resist loss of protein sample.
- Western ReProbe™ and Western ReProbe™ PLUS differ in harshness of treatment. First, try the gentler Western ReProbe™ and check for efficiency by incubating with chemiluminescent substrate. If you still see bands, then you need to strip for longer or use the harsher Western ReProbe™ PLUS. After checking efficiency, the membrane must be rinsed several times with buffer and then blocked before proceeding to reprobe with antibodies.
- Double check your target proteins remain on the membrane. The stripping procedure removes some sample protein from the membrane so it is not advisable to make quantitative comparisons of target protein abundance before and after stripping. Test the effects of your stripping protocol on your bound target protein by comparing your blot’s signal strength (with a specific antibody) both before and after stripping. If a drop in signal strength occurs, then perform shorter incubations or use a gentler stripping method.
- Check that your detection method is compatible with stripping. Stripping methods (including with Western ReProbe™ and Western ReProbe™ PLUS) are not recommended for stripping color producing western blot detection methods that use substrates such as TMB, chloronapthol and DAB. These substrates form a physical precipitate on the membrane which is different than the antibody. Therefore, dissociation of your antibodies will not remove the colored bands.
- Keep your blot wet. Antibodies can permanently bind to your membrane if your blot dries out any time after transfer.
- Remember to re-block. All stripping methods cause dissociation of your blocking proteins as well as your antibodies. Failure to re-block will result in a black blot, but the membrane can still be stripped, reprobed and re-blocked.
For further tips and tricks for Western blotting review: