Antibodies 101: Stripping and Reprobing Western Blots

By Emily P. Bentley

Emily P. Bentley August 01, 2024

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!

Before you begin

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.

Preparing your western blot

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!

Stripping your membrane

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.

Mild stripping

The mild stripping solution uses low pH to disrupt antibody binding to antigens.

Ingredient	Concentration or %
Glycine or Glycine-HCl	25–200 mM
SDS	0.1%–1% (w/v)
Tween 20 (optional)	1% (v/v)
HCl	Adjust to pH 2.0–2.2

For the mildest stripping solution, omit the Tween 20.

Once your solution is made, you’ll want to:

Rinse the membrane in water or fresh buffer.
Cover the membrane in stripping solution and agitate for 5–30 minutes at room temperature. The incubation time will depend on your blot — antibodies that bind more strongly or that have saturated the blot may need longer incubation for effective stripping. Some blots benefit from an additional 5–10 minutes of incubation at 37 °C.
Wash the membrane for 5–10 minutes 2–3x in fresh wash buffer such as PBS or TBST.

Stringent stripping

The stringent stripping solution uses heat and detergent to denature and remove antibodies but is also likely to remove more of your target protein.

Ingredient	Concentration or %	Purpose
Tris HCl	62.5 mM	Buffer
SDS	2% (w/v)	Detergent
Β-mercaptoethanol (βME)	100–115 mM = 0.7%–0.8% (v/v)	Breaks disulfide bonds
HCl	Adjust pH to 6.7–6.8	Adjust pH

Pro tip! βME is a reducing agent and gets oxidized over time, so it should always be added fresh to solutions. It is also volatile and toxic (and smells bad!), so be sure to use it under a fume hood.

Rinse the membrane in water or fresh buffer.
Cover the membrane in stripping solution and agitate for 30–45 minutes at 50 °C.
Wash the membrane for 5–10 minutes 2–6x in fresh wash buffer such as PBS or TBST. Note that βME reacts with antibodies, so thorough washing is critical.

Assess the stripping

Now it’s time to check to make sure the antibodies from your first round of detection are actually gone. First, simply repeat the imaging step to check if any secondary antibody remains, whether that involves adding fresh chemiluminescent substrate or imaging your fluorescent probe. Then, if you don’t see any signal, you’ll want to check for the primary antibody by re-blocking your membrane and re-incubating with your secondary antibody. This is the one time in western blotting that you’re looking for a “blank” result!

If some signal remains, you may want to repeat your stripping procedure, perhaps using longer incubation times, or try the stringent solution if you started with the mild one.

Reprobe

Once your membrane is clean of antibody, you’re ready to repeat your western blot as normal. Be sure to rinse away the secondary antibody you used to assess the stripping, and don’t forget to re-block your membrane!

Depending on the system, you may be able to strip and reprobe your western blot several times — some researchers report up to ten cycles! Just remember that a bit of sample is lost in each round of stripping, so it pays to plan ahead.