Understanding the Western Blotting Technique

What’s the name of that protein? It’s a common question posed by researchers in the laboratory environment. To detect and identify a single protein, researchers head to the lab, where they use a test called the western blot to identify a mysterious protein molecule from amongst a mixture of various proteins.

Origin of Western Blotting

First, you should know that despite its name, the term “western” is lowercase when used to describe western blotting. The spelling has to do with the history of this widely used laboratory test.

In 1975, inventor Edwin Southern came up with a new way to analyze DNA for its identity, how prevalent it is in a particular sample, and its size.

He based his self-named “Southern blot” technique on electrophoresis, which is a method to separate fragments of DNA according to their size. The fragments were transferred to a membrane, which Southern then hybridized using DNA labeled with a radioactive substance (to help scientists track the material during an experiment). Using a radio-labeled DNA probe, Southern identified a specific sequence of DNA from the sample.

Two years, Stanford University researchers created a similar test used to identify RNA molecules inside a sample of RNA, and they gave this technique the name “northern blot.”

Fast-forwarding to 1979, a post-doctoral researcher named W. Neal Burnette was inspired by the northern blot technique while working at Fred Hutchinson Cancer Research Center in Washington State.

He was stymied in his attempts to identify certain antigens in a sample. The polyacrylamide gels used in the northern blot approach would not let him visualize interactions between proteins and antibodies. So, Burnette made a solid-phase replica of the gel to blot proteins onto nitrocellulose paper (instead of the traditional paper modified by chemicals used in the northern blot).

Burnette announced his method in 1979 by submitting a paper to Analytical Biochemistry, which rejected the manuscript because the editors were displeased with the naming convention of “western blot” following the other blot methods. But as scientists heard about the western blot method and started using it, support grew for this new approach to identifying proteins in the lab.

Western Blotting Basics

Researchers in laboratories can answer nagging questions about a specific protein found in a mixture thanks to western blotting.

Work begins by preparing the sample of proteins, mixing it with sodium dodecyl sulfate, which unfolds the proteins into chains that line up, and gives them a negative electric charge, according to Nature.

Then, gel electrophoresis separates these chains of proteins by size. They transfer from the gel to a blotting membrane. At this point, the membrane with protein bands is treated to block activity, which keeps reactions from happening.

The researcher places the membrane into an incubator along with what’s termed the primary antibody, which is used here to help find the protein the researcher is searching for in the sample.

Once incubation has finished, any remnants of the primary antibody not bound to target protein molecules are washed away.

At this point, the researcher incubates the membrane once more, but now uses a second antibody connected to a “reporter enzyme” to produce a specific light or color to allow researchers to see how much of the targeted protein is actually in the sample.

The Western Blot Is a Mainstay of Modern Science and Research

From conducting medical research to diagnosing an ailment, western blotting is a powerful workhorse in the laboratory.

Indeed, anyone working in a lab to analyze samples or conduct research that depends on identifying specific proteins will make frequent use of the western blot. Understanding the basics of western blotting helps put this vital data into perspective.

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