Stripping and Western Blotting Part 2: Factors to Consider

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This is part II of our “Stripping and Western Blotting” series, so check out part I if you haven’t yet.

In the last blog, I covered the ins and outs of using stripping buffer. Now that you know which kind of stripping buffer to use and when, let’s go over when you’d want to strip and reprobe your Western blot.

One of the benefits of Western blotting is the ability to probe one membrane for multiple proteins, which gives researchers more data from one experiment, called multiplexing. Multiplexing (Figure 1) is often done by probing the same membrane for multiple targets at the same time; however, it is not always possible to probe for all targets at once. In these cases, the membrane must go through two rounds of detection. In the first round, after imaging, the membrane needs to be stripped of the original primary and secondary antibodies. During the second round, the membrane is ready to be reprobed with a different primary and secondary antibody.

Multiplex fluorescent Western blot from Azure Biosystems imager
Figure 1. Digital images of 4-color multiplex Western Blot. Using distinct fluorescent and near-infrared targeting antibodies can detect each wavelength and merge them into a four-color multiplex image. No background noise or bleeding between channels. Image captured with the Sapphire Biomolecular Imager

When to strip and reprobe your Western blot

Repeating the entire Western blot process requires a lot of time, sample, and reagents. If you strip the membrane after imaging and reprobe it, you can detect additional proteins without repeating your experiment. Whether it is necessary to strip the membrane and reprobe depends on the specific experiment and situation.

Let’s look at the instances where you would strip and reprobe a Western blot membrane:

You want to look at proteins of similar molecular weight

If you are probing sequentially for proteins of a similar size, residual bands from the first protein may confound the results of the second protein. To avoid any confusion in the data, stripping the membrane removes residual primary or secondary antibodies from the first blot.

You want to avoid background noise

Even if the sample proteins are not similar in molecular weight, they could still affect the results of subsequent blots. For instance, using antibodies of the same species for proteins being detected sequentially could cause background bands to show up. During the second round of probing, using the same species-specific secondary could result in the bands from the first antibody detection round appearing.

Stripping the membrane removes the primary antibody used for detection of the first protein and prevents them from showing up in subsequent blots.  

You want to determine the relative abundance of proteins

Due to the nature of stripping, quantitative analysis is not recommended, but the relative abundance of proteins may be reliably compared.

You want to evaluate loading control proteins

Loading control proteins are used to assess relative amounts of unknown samples, which leads to the potential for higher numbers of antibody pairs when comparing loading controls to unknowns. Stripping and reprobing proves a great option in this situation.

You need to correct an error (such as using the wrong antibody)

If an accident occurs and the wrong primary antibody is added, stripping the membrane allows the experiment to be salvaged. This can save both time and resources, compared to repeating the entire experiment.

You need to fix other mistakes

A number of issues can occur throughout the Western blotting process. Some of these can be addressed by stripping and reprobing, instead of scrapping the entire experiment.

For example, if it is determined the blocking time needs to be extended, strip the antibodies, adjust the blocking time, and reprobe.

There are many steps that need optimization in the Western blotting process. Blocking reagent, primary and secondary antibody concentrations, and incubation times all contribute to the final quality of the blot. Stripping the membrane supports this optimization process by using the same membrane to determine the best conditions.

Two scientists looking at multiplex fluorescent Western blot on Azure 600 Western blot imager

If you’re looking for a reliable imager to image your Western blots, your search ends here. Azure Imagers are high performance Western blot imaging systems capable of NIR fluorescence, visible fluorescence, and chemiluminescence. Request a free, virtual demo of an Azure Imaging System, and say “Hello!” to beautiful Western blots.

quantitative western blot basics


Get a quick overview of the steps you can take to ensure your Western blots are quantitative. This free guide also includes a troubleshooting section and tear-out quantitative Western blotting checklist.

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