How Scientists Troubleshoot Issues With Western Blotting

When it comes to identifying proteins in a blood sample, doctors, researchers, and other scientists often rely on the western blot technique. People routinely order western blotting tests to help with diagnosing diseases as well as to track how proteins are expressed during experiments.

Overview of the Western Blot

The western blot is a technique developed by W. Neal Burnette back in 1979. He needed a way to visualize how antibodies and proteins interact. Burnette, working out of Washington State’s Fred Hutchinson Cancer Research Center, based his “western blot” on earlier pioneering work with electrophoresis, which originated as the Southern blot (from the work of Edwin Southern).

You employ western blotting to identify targeted strands of genetic sequences, which are found with the help of a radio-labeled probe made of DNA.

It allows you to find specific proteins and see how many of them there are in a given sample. The western blot makes possible a wide range of applications for finding and measuring protein molecules.

Using Western Blotting in a Laboratory Environment

Western blotting involves separating proteins from a given sample using gel electrophoresis. This serves to organize them according to factors such as their weight or electrical charge. Then, the researcher will transfer the sorted proteins from their gel to a membrane.

At this point, to visualize the proteins you’ve targeted, you use primary antibodies to stain the membrane, along with secondary antibodies that themselves adhere to the primary antibodies.

Troubleshooting Problems With the Western Blot

In a laboratory, experiments do not always go as smoothly as researchers would prefer. As noted by Before It’s News, “Challenges can arise at any point during the western blotting process, which has a seemingly random tendency to fail.”

There are five main types of challenges with western blotting. To address them, scientists have developed methods to troubleshoot the western blot:

* Bands You Didn’t Expect: Typically the result of the protease degrading, you will find bands in places they shouldn’t normally appear. The response is to adjust the antibody you’re using and then try again using a fresh sample.

* Zero Bands: This is a bit more complicated to troubleshoot since the issue could be with the antigen, antibody, or the buffer. Failure to use the correct secondary or primary antibody can cause zero bands in results. Use an antigen from another project to see if the problem has to do with your sample or with an aspect of the test.

* Bands Are Faint: If the antibody isn’t available in high enough concentration during the analysis, you might wind up with a set of faint bands. One response is to add time to the exposure period. Or, employ bovine serum albumin instead of the non-fat dry milk typically used in labs (because this dairy product can obscure antigens).

* Blot Background is High: An excessive level of antibodies can cause the blot to have a high background, binding to the membrane. Or, your problem could be the result of the buffer being old. In some cases, high background comes when the exposure time is too great. Try a shorter exposure time.

* Uneven Spots on the Sample: Trapped air bubbles make a sample have unequal spots, making dark areas on the film and confusing your ability to identify protein molecules. The solution is to wash the background and refrain from agitating the material unevenly.

Troubleshooting the Western Blot for a More Productive Laboratory Effort

Despite the fact that researchers need to be prepared to troubleshoot random, unexpected problems during experiments involving the western blot, it is still a valuable technique. It’s safe to say that scientists will continue to come up with ways to work around these issues, such as with automated systems.

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