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April 27, 2018

Stripping and Reprobing Western Blot Membrane: Problems and Solutions

By GE Healthcare Life Sciences

In this post, we discuss ways of maximizing your Western blot membrane use by stripping and reprobing with another primary antibody. You can use this technique to detect more than one protein molecule on the same membrane, to conserve resources, reduce cost and save valuable time.

Stripping a Western Blotting Membrane

Stripping the Western blotting membrane allows you to use it multiple times for the same set of samples. Whichever membrane stripping method you use, you can remove the primary and secondary antibodies and probe the membrane again.

Multiple uses of your blotting membrane can be especially useful if your proteins of interest are only available in limited quantities. For example, this might be because your protein molecule is difficult to purify, or only present at low concentrations in cell extracts. Or perhaps, you may only have a limited amount of cell extract. Regardless of the reason, you might only have enough protein for one Western blot. On the other hand, your experiment might depend on visualizing two protein molecules of similar molecular weight or a loading control.

For a detailed look at stripping and reprobing methods, see our in-depth article on the subject, or if you are looking for some quick hints and tips and an easy how-to guide then read on below!

How to Reprobe a Western Blotting Membrane

There are a few things to bear in mind once you know you are going to reuse a membrane.

Your target protein abundance and antibody affinities are two points to consider. These properties influence your membrane stripping effectiveness, and which antibody you use first.

We have outlined four different strategies below. The one you use depends on your specific experimental circumstances, namely your protein abundance and antibody affinity. Try matching your problems to the solutions and you will be on your way to a successful stripping protocol.

Strategy 1
Problem: You have two proteins of similar abundance two antibodies of similar affinity.

Solution: You can detect either protein first, strip the membrane, and then detect the remaining protein.

Strategy 2
Problem: You have two proteins of similar abundance two antibodies of unequal affinity.

Solution: Detect the protein with the lowest affinity antibody first, strip the membrane, and then detect the protein with the highest affinity antibody.

Strategy 3
Problem: You have two proteins of different abundances (one high and one low) and antibodies of equal affinity.

Solution: Detect the low-abundance protein first, strip the membrane, and then detect the high-abundance protein.

Strategy 4
Problem: You have two proteins of different abundances (one high and one low) and antibodies of unequal affinity.

Solution: Detect the low-abundance protein first, strip the membrane, and then detect the high-abundance protein

How to Strip a Western Blotting Membrane

Once you have defined your strategy, there are a few methods available for stripping your Western blotting membranes. Table 1 summarizes the different membrane stripping options. Try the heat and detergent method first. If this is not successful, try one or more of the other methods listed in the table.

Table 1.Summary of Western blot membrane stripping options

Stripping method Stripping buffer Protocol Notes
Heat and detergent 2% SDS + 100 mM β-mercaptoethanol in 62.5 mM Tris-HCl, pH 6.8 Incubate the membrane with stripping buffer for 30 min at 50°C to 70°C. Optimal temperature might vary. Test different conditions. Find more informationon optimizing conditions.
Low pH 25 mM glycine-HCl, pH 2.0, supplemented with 1% SDS Soak the membranes in stripping buffer for 30 min under constant agitation. A milder protocol for chemiluminescence applications:Incubation in 0.2 M glycine (pH 2.8) at room temperature for 30 min, then two washes in wash buffer for 10 min each.
High pH 0.2 M NaOH Incubate the membrane twice in stripping buffer for 5 min, then wash in water for 5 min. It might be necessary to optimize NaOH concentration and incubation time.
Might require reblocking.
High salt solution PBS or TBS buffer supplemented with 0.5 M NaCl and 0.2% SDS Incubate the membrane for 30 to120 min, and then rinse with water. Might require reblocking.

SDS: Sodium dodecyl sulfate; RT: Room Temperature; PBS: Phosphate buffered saline; TBS: Tris buffered saline

Download the Western blotting principles and methods handbook  to learn how to go about stripping and reprobing.