Understanding Western Blotting, Step-by-Step

Before a scientist, researcher or technician can understand what kind of proteins is in a sample that they are dealing with, tests have to be performed. In many cases, the preferred technique to identify protein molecules and tell them apart from one another is western blotting.

Read on for insight into the nature of western blotting, why people use this technique, and what steps are involved in a western blot procedure.

What Is Western Blotting?

A bit of a history lesson is in order. Back in 1975, an inventor named Edwin Southern developed a method to learn what DNA is in a sample, as well as how much of the DNA is present and the size of the sequence.

As is often the case with inventors, Southern named the technique after himself, giving the term “Southern blot” to a process that harnesses electrophoresis to separate DNA fragments by size, which is essential in learning what you are working with.

Later, researchers at Stanford University developed a related technique, in this case, to identify molecules of RNA in a given sample. The name of this version is the “northern blot.” In 1979, the procedure was advanced again, this time by W. Neal Burnette, as noted by the American Society for Biochemistry and Molecular Biology.

He was using the northern blot at the Fred Hutchinson Cancer Research Center, located in Washington State. But the northern blot wouldn’t help him determine specific antigens in a lab because of the nature of the polyacrylamide gels that technicians rely on in northern blots.

By using a solid-phase version of the gel, Burnette could finally visualize interactions of antibodies and target proteins. He dubbed his method “western blotting” in deference to its origin as the southern and northern blots.

What Is the Purpose of Western Blotting?

The reason scientists use western blotting is to understand what particular proteins are in a given sample. This is useful when you want to see how different proteins react when a human or animal has a certain disease.

Or, you want to learn what a particular drug’s effect has on a person or on the course of a disease. You also employ western blots to identify in a clinic if a person has an ailment. Doctors will order lab tests to rule out if a patient has HIV, for example.

Many centers are using the western blot to identify clues in the ongoing global novel coronavirus pandemic that’s been causing high rates of infection of COVID-19. Discovering variants quicker in the coronavirus helps medical professionals identify mutations, to classify, study and determine how they might adjust vaccines to account for the changes in the virus itself.

Crucial Steps in Western Blotting

Scientists follow six basic steps when using the western blot, according to Creative Proteomics. The steps are:

  1. Prepare the sample after extracting it from a source, such as a collection of cells or some tissue.
  2. Apply gel electrophoresis. This moves the proteins under electrical stimulation to separate them into different proteins according to molecular weight.
  3. Transfer the proteins from the gel to a solid membrane to support them during the next step.
  4. Employ blocking to keep antibodies from randomly binding to the support membrane. Often, the researchers will use non-fat dry milk to stop such binding.
  5. Incubate the antibodies. A scientist or technician will select a primary antibody to try to detect an antigen in the sample. Rinsing the membrane and then exposing it to a secondary antibody.
  6. Detect and visualize the protein under study. You use an enzyme to react with the substrate, resulting in a colored material that helps identify how dense the proteins are and where they are.

Western Blotting Is a Powerful Mainstay of Labs and Clinics

With western blotting being such a powerful tool to identify mysterious proteins in a sample and to determine how much of a particular protein appears, it’s no wonder that so many scientists and research institutions have come to rely on the procedure in their daily routines.

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