Stripping and Western Blotting Part 1: How Stripping Buffer Works

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How many times can you strip a Western blot?

Stripping buffer works by removing the primary and secondary antibodies from the membrane. Each time you use stripping buffer, you remove a bit of the protein sample too. The number of times a membrane can be stripped and reprobed for Western blotting depends on the stripping protocol you choose and how much of the protein of interest is present. Keep in mind that eventually, the amount of the protein sample lost will be outside the minimum threshold for detection.

With that being said, if a gentle stripping protocol is applied, the process can be repeated more times than with a harsh stripping protocol. In general, a membrane can be stripped about 3 times, though some have reported stripping a membrane up to 10 times. The number of times will depend on a number of factors and can only be determined with testing.

You should always aim to set up your experiment to probe for the lowest expressed protein first. It’s easier to strip smaller amounts of antibody than larger amounts. This will be a two-part blog series. In part one, I am going to cover the main types of stripping methods. Stay tuned for part two!

The three main types of stripping methods we are going to cover in part I are: mild, harsh, and commercial. Let’s go over how long each method should take and what each entails.

Time needed: approximately 20 minutes

The mild stripping method uses a low pH glycine solution to dissociate the antibodies on the membrane from their target proteins. It does this by altering the binding site of the antibodies enough to render them inactive. 

Time needed: approximately 30-45 minutes

This method uses Tris-HCl, SDS, and a reducing agent like beta-mercaptoethanol

Time needed: If you use HRP Stripping buffer from Azure Biosystems, it only takes 5 minutes to strip the antibodies from the membrane.

Commercially available stripping buffers make the stripping process quick and easy.

Choosing the right stripping buffer

Various factors go into determining which stripping buffer is best suited for any set of conditions. For example, if the protein of interest gives a strong signal, you may want to consider starting with a harsh stripping method. If it is desirable to reuse the primary antibodies after effectively detaching them from the membrane, using a mild stripping method would be ideal.

If it is not clear which stripping buffer to choose, consider testing a mild stripping buffer first and switch to a harsher method if this is unsuccessful. 

After using a stripping buffer, test its effectiveness by probing with a secondary antibody alone and using chemiluminescent substrates to detect any signal. If the stripping process was successful, no signal should be detected. 

In the part II of Stripping and Western Blotting, I’ll discuss the scenarios where you’d want to strip and reprobe.

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SOURCES

  1. Davis, S. “Western Blot Membrane Stripping.” St John’s Laboratory, 12 November 2020, https://stjohnslabs.com/blog/western-blot-membrane-stripping. Accessed 24 September 2022.

  2. van Geldermalsen, Michelle. “Get your stripping stripes! Find out how to strip and re-blot your Western.” Bitesize Bio, 5 August 2014, https://bitesizebio.com/20884/get-your-stripping-stripes-find-out-how-to-strip-and-re-blot-your-western/. Accessed 24 September 2022. 

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