Western blots are a great tool to identify a protein of interest in a complicated solution like cell lysate. But they can be a lot of work — and what if you want to detect more than one protein in your sample? Or what if something weird happened during your western and your results look… funky? Do you need to start a whole new blot?
Never fear: membrane stripping is here!
Rather than start over, you can remove the detection antibodies from your membrane, allowing you to probe the blot again, almost like new. This approach preserves your sample and saves you from having to run a whole new gel and membrane transfer. Let’s get started!
In the best case, you know ahead of time that you want to probe your blot with multiple antibodies. You’ll want to set up your blot with a polyvinylidene difluoride (PVDF) membrane, which is sturdier and retains the blotted protein better than a nitrocellulose membrane.
If you have already run your blot using nitrocellulose, you may decide you have nothing to lose by trying to strip and reprobe. In this case, we recommend using mild stripping solution (see below) to minimize loss of your sample from the membrane.
Next, plan out the order of targets you plan to probe for. Even using PVDF, some of your target protein will be removed from the membrane along with the antibodies, especially with more stringent stripping methods. If you are planning to detect multiple proteins through several rounds of stripping and reprobing, maximize your signal by starting with the least-abundant protein and working toward the most abundant. If the abundance of your targets isn’t too different, consider antibody affinity: stronger binding antibodies will require harsher stripping to remove, so start with the weaker binding ones first to preserve your sample.
Pro tip! Because some protein is lost each time you strip a blot, you should not compare the strength of bands from different rounds of detection.
Finally, stripping and reprobing works best with western blots that use chemiluminescent or fluorescent detection, like horseradish peroxidase (HRP) or Alexa Fluor dyes, respectively. Chromogenic detection produces colored precipitates that permanently stain the membrane, making that section of the blot unusable for reprobing.
Run your gel and membrane transfer as normal. Immediately after transfer, dry the membrane to maximize protein retention.
Following this step, most PVDF membranes recommend re-wetting with methanol before rinsing with wash buffer, as dry PVDF won’t evenly re-absorb aqueous buffer. Be sure to check the manufacturer’s instructions for the membrane you are using.
Dry nitrocellulose membranes are brittle, so proceed with caution. Unlike PVDF, they can be returned directly to buffer without a re-wetting step.
Important: do not dry your membrane with antibodies on it if you plan to reprobe! Just as your sample protein is retained better, antibodies will stick permanently to a membrane that has been dried, no matter what stripping methods you throw at it. Ideally, dry your membrane once before any detection steps to avoid retaining residual antibodies. If you have already applied antibodies and need to dry the membrane for storage, but plan to reprobe it later, strip it thoroughly first.
Then continue on with your western blot as normal!
Alright, so you’ve dried your membrane and returned it to buffer, you’ve run and imaged your western blot with your first target, and now you’re ready for round two (even if round two is just another attempt at the same target!). In the stripping step, you’ll remove the primary and secondary antibodies from your membrane, clearing the way for the second blot. There are multiple ways to strip blots, and the best choice will depend on your specific experiment.
Remember, stripping your membrane will remove some of your sample —and harsher stripping means more sample loss. If you are still optimizing your system, start with a mild stripping solution and move onto a stringent solution if the mild is not effective.
You may choose to make your own stripping solution using a recipe like the ones shared by Bio-Techne, ThermoFisher, or Millipore Sigma. We’ll share the basic composition of these solutions below. Alternatively, companies including ThermoFisher, Millipore Sigma, and Azure Biosystems offer pre-made stripping solutions or kits.
The mild stripping solution uses low pH to disrupt antibody binding to antigens.