Over 40 Years with Western Blotting

Molecular biology is known for its complexity, posing challenges to scientists since before Rockefeller Foundation Natural Sciences Director Warren Weaver introduced the term to the world in 1938. Identifying what happens within organisms at the cellular level – and smaller – is a fascinating discipline requiring ever-evolving techniques.

One such stratagem is western blotting. Today, this method represents more than four decades of success in the fields of molecular biology, biotechnology, immunogenetics and proteomics, which refers to “the large-scale characterization of the entire protein complement of a cell line, tissue, or organism.”

Let’s take a quick look at its history, evolution and continued importance today.

What Is Western Blotting?

Often credited to W. Neal Burnette, who first described it in 1980, this method is based on an evolved form of southern blotting and its close cousin northern blotting. As Burnette’s lab was based on the west coast, it was a reasonable name that brought the new science into the fold but distinguished it from its precursors.

(As is so often the case, the true origins of the method are disputed. However, most credit this technique to Burnette because his approach was simpler than those of his contemporaries.)

Also called the western blot, protein blotting and immunoblotting, this technique involves the use of gels and antibodies to transfer proteins from a mixture extracted from inside cells. As a study published in the North American Journal of Medicine & Science explains, researchers are able to extract specific proteins from complex mixtures of them, majorly simplifying the task of isolating proteins of interest.

How Does It Work?

The western blot follows a series of specific steps to isolate the desired protein from the background mixture. These are as follows:

  1. Electrophoresis: Gel electrophoresis allows researchers to separate the sample’s proteins by size. Running a current through the gel affects the proteins differently based on their innate charge, causing them to move at different rates and thus separate out.
  2. Membrane Transfer: Researchers then move the separated proteins to what’s called a “blotting membrane,” then introduce an antibody specific to the protein of interest.
  3. Blocking Buffer Application: To prevent the antibody actually binding to the protein and therefore impacting the sample, researchers use a blocking buffer.
  4. Antibody Binding: Once that is applied, they then allow the antibody to bind to the protein.
  5. Blot Washing: A quick bath removes any antibodies that have not bound to the protein to clean the sample.
  6. Secondary Antibody Binding: Now scientists introduce a second antibody that will bind to the primary antibody, this one treated to reveal the location of the target protein.
  7. Blot Washing, Redux: The blot is again washed to remove excess secondary antibodies.
  8. Protein Detection: Finally, the sample is ready. Researchers can now detect the protein using their method of choice – colorimetric, luminescent, fluorescent-based on the type of secondary antibody they used.

What Is Western Blotting Used For?

Today, this originally overlooked method is hugely consequently in a variety of research fields, including medical, pharmaceutical and immunological. It can be used to determine whether, for example:

  • Proteins are expressed normally in cells
  • They are smaller (meaning they’re breaking down for some reason) or larger (meaning they have been inflated for some reason)
  • Various tissues express proteins at different rates
  • Proteins change in response to a particular disease
  • A specific drug has an effect on a specific protein of interest

… and more. Today, this technique is critical to medical science and will continue to be for the next 40 years as well. That’s worth celebrating!

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