Western Blot Membrane Stripping: A Guide to Removing Antibodies and Re-Staining

Western blots are a powerful tool for detecting specific proteins in a sample, but sometimes you may need to remove antibodies from the membrane to re-stain with different probes. This process is known as blot off 'blot off', and it can be a crucial step in your research. In this article, we will explore the techniques involved in stripping and re-staining western blots.

Why Stripping is Necessary

There are several reasons why you may need to strip a western blot membrane. For instance, if you want to investigate more than one protein on the same blot, you may need to remove the primary antibody from the membrane before adding a new probe. Additionally, stripping can be useful when you want to check for the presence or absence of a specific protein.

How to Strip a Western Blot Membrane

Stripping involves removing the primary and secondary antibodies from the western blot membrane using a chemical treatment. The process typically starts with incubating the membrane in a volume of buffer that will cover it, followed by rinsing several times with distilled water. Some researchers report successfully staining a membrane after stripping ten or more times.

The efficiency of stripping can be checked by incubating the membrane with chemiluminescent detection reagent. This step can be repeated for probing with several antibodies, though the potential signal may be weaker and the background higher after each round of stripping.

Choosing the Right Reagents

When selecting a stripping protocol, it is essential to choose the right reagents. Chemiluminescent reagents such as ECL are recommended because they will not leave a stain on the membrane and are more sensitive than colorimetric reagents. Colorimetric/chromogenic detection reagents, on the other hand, can leave a permanent visible stain on the membrane that may interfere with subsequent detection of targets.

Tips for Successful Stripping

When stripping a western blot membrane, it is crucial to follow the right protocol. Here are some tips to keep in mind:

* Use a PVDF membrane to minimize loss of sample protein.

* Avoid making quantitative comparisons of targets probed before and after stripping since the procedure removes some sample protein from the membrane.

* When probing for multiple targets, consider stripping and re-probing a single membrane instead of running and blotting multiple gels.

In conclusion, blot off 'blot off' is an essential step in western blots that allows researchers to remove antibodies from the membrane and re-stain with different probes. By following the right protocol and choosing the right reagents, you can successfully strip your western blot membranes and achieve the desired results. Remember to always follow proper techniques and guidelines when working with western blots to ensure accurate and reliable results.

Blot off