BioTechniques explains how researchers use western blotting, a key technique in biochemistry, to detect proteins.

Western blotting, also known as immunoblotting, is a biochemical technique that provides crucial information about proteins in complex samples. This is useful in many scientific and medical fields, including cancer care and pain management. By using a western blot, researchers can detect specific proteins in a complex mixture of proteins that have been extracted from cells. As a result, the western blot is core to many protein studies. In fact, 9% of all protein-related papers cite the technique.

Since western blotting's inception approximately 40 years ago, the technique has grown in popularity. Today, almost every biochemistry lab in the world uses the western blot, making the most of its cost-effectiveness and simplicity.

The leading life sciences journal BioTechniques offers a huge amount of information on the western blot in both its print journal and its online media. This content provides key insights into the western blot's biochemical principles, clinical significance, and the latest developments that troubleshoot its limitations.

When Is Western Blotting Used?

Scientists use the western blot in a variety of applications, from basic research to diagnosing diseases. For example, patients can take a western blot test to check for HIV. After taking a small blood sample to detect HIV antibodies, it's then possible to perform a western blot test to separate the blood proteins and detect any HIV antibodies that indicate an infection.

Western blotting is often helpful in the early stages of studies. Researchers use the technique to:

  • Detect specific proteins in samples
  • Determine rudimentary information about a protein's size
  • Validate the successful transfection of a cell colony with a non-viral vector.

Researchers also sometimes use the western blot later in studies, often to confirm the success of a gene-editing technique like CRISPR.

How Does Western Blotting Work?

Western blotting identifies the molecular weight, quantity, and post-translational modifications of proteins. It is therefore a powerful technique when it comes to monitoring changes - such as expression and modifications - in proteins.

There are five stages in the process.

1.  Sample Preparation

Researchers prepare the sample, which could be tissue, cells, or another solution. Sometimes, they break down the sample through a process like blending, sonication, or homogenisation. Researchers add buffers to lyse the sample, make the target proteins available, and give them a negative charge.

2.  Protein Separation

Next, researchers separate the proteins in a sample through gel electrophoresis. This technique separates charged molecules according to their physical properties by forcing protein or DNA through a gel by an electrical current.

Researchers might separate the proteins by several factors, like molecular weight, electric charge, or isoelectric point. This depends on the nature of the gel and the treatment of the sample.

The most common type of electrophoresis is SDS-PAGE, which uses polyacrylamide gels and sodium dodecyl sulfate buffers. This process results in denatured proteins, each separated according to molecular mass.

3.  Blotting

Once researchers have separated the proteins, they need to make these proteins accessible to antibody detection. Researchers achieve this by transferring (blotting) the proteins from the gel onto a membrane - usually nitrocellulose or polyvinylidene difluoride (PVDF).

There are lots of transfer methods available, including capillary transfer, diffusion transfer, and vacuum blotting transfer. However, the most common transfer process is electroblotting, which is quicker and more efficient than many alternatives. Electroblotting uses an electric current to pull the proteins from gel to membrane. Electroblotting doesn't disrupt the organisation that the proteins have in the gel.

4.  Antibody Incubation

Then, researchers 'block' the membrane to avoid non-specific antibody binding. They use blocking buffers like milk, serum, or highly purified proteins. The blocking agent improves the sensitivity of the assay by minimising background interference, which improves the signal-to-noise ratio. Different blocking buffers are better suited to different western blotting tests.

Researchers then stain the membrane with primary antibodies that are specific to the target protein. The type of primary antibody depends on the antigen that researchers are trying to detect. Usually, the primary antibody that recognises the target protein isn't directly detectable.

Next, researchers wash the excess primary antibody off and add a tagged secondary antibody, which binds to the primary antibody. It's the tagged secondary antibody that ultimately detects the target antigen. Many secondary antibodies are suitable for western blot detection. The type used depends on any tags linked to the primary antibody and the species of animal in which the primary antibody was raised. Researchers can use several methods to visualise the tagged antibody. For example, immunofluorescence enables researchers to indirectly detect a target protein.

5.  Imaging and Data Analysis

Finally, researchers detect the reporter molecule of the secondary antibody. This allows them to establish the presence and quantity of the target protein in the original sample. They may use a film or CCD camera or scanner during the imaging process. Then, during analysis, researchers confirm the presence, amount, and size of a protein.

What Are the Challenges Associated With Western Blotting?

Although the western blot is widespread, challenges can arise at every stage of the process. It doesn't matter how experienced researchers are - the western blot has a seemingly random tendency to fail. However, new technologies that show researchers where issues are arising are now emerging.

For example, Bio-Rad's Stain-Free Western Blotting technology eliminates the need to stain gels and membranes. The automated system tells researchers that they have completed key steps, such as electrophoresis and blotting, successfully. The technology avoids researchers applying expensive antibodies and detection agents onto already failed blots, saving time and money.

Where Does Western Blotting Come From?

The name 'western blot' is a play on the 'Southern blot', a technique coined by Edwin Southern in 1975. Southern blotting uses electrophoresis to separate DNA fragments based on size. Researchers can then transfer the DNA to a membrane and complete a hybridisation process using a radiolabelled DNA probe. This way, researchers can detect a specific DNA sequence in a sample and analyse the identity, size, and abundance of this DNA.

In 1977, James Alwin, David Kemp, and George Stark, scientists from Stanford University (CA, USA), came up with a similar technique: northern blotting. This technique also uses a radiolabelled DNA probe but, instead of detecting DNA sequences, detects a specific RNA molecule in an RNA sample.

In 1979, W Neal Burnette, a postdoctoral researcher who was working at the Fred Hutchinson Cancer Research Center (WA, USA), tried to detect specific antigens in a protein mixture. He attempted to combine SDS-PAGE electrophoresis with a radioimmunoassay, but he couldn't visualise the interactions between the separated proteins and antibodies in polyacrylamide gels.

Instead, Burnette drew inspiration from northern blotting and produced a solid-phase replica of the gel. He found that electrophoresis enabled the blotting of proteins from SDS-PAGE gels onto nitrocellulose paper and that this worked better than chemically modified paper. Burnette then used [125I] -labelled protein A, which binds most antibody-antigen complexes, removing the need for a second radiolabelled antibody. By blocking non-specific binding sites of the protein A and the antibody to nitrocellulose paper, Burnette was able to obtain clear radiographic images of antibody-specific antigens.

Though named after the Southern blot, the western blot's name also refers to the U.S. west coast location of the lab where Burnette came up with the technique.

In 1979, Analytical Biochemistry rejected Burnette's submission of the western blot methodology, largely because of its naming convention. Nonetheless, the technique has since been popularised by other scientists. And 2019 marked the western blot's 40th birthday, which life science organisations around the world - including BioTechniques - celebrated.

Other Blotting Methods

Since the inception of the western blot in 1981, scientists have created several other blotting methods. One example is eastern blotting, which involves detecting post-translational protein modification. Researchers can achieve this by using substrates to identify lipoylation, glycosylation, and phosphorylation. Variations of this technique include far eastern blotting, middle eastern blotting, and eastern western blotting.

Another recent blotting technique is southwestern blotting, which combines elements of the western and Southern blots. This process involves identifying and characterising DNA-binding proteins using DNA probes (like in Southern blotting) and protein blotting (like in western blotting). Although the southwestern blotting method was published before the western blotting method, the technique still alludes to the western blot's name, which is indicative of its popularity.

Who Takes Credit for the Blot?

There is some controversy over who should take credit for inventing the blot. This is because while Burnette was developing the western blot, Harry Towbin and his colleagues at the Friedrich Miescher Institute (Switzerland) were crafting a similar technique, also inspired by the northern blot. Like western blotting, this technique involved the electrophoretic transfer of proteins from SDS-PAGE gels to a membrane and the use of antibodies for detection.

Meanwhile, Stark and his colleagues at Stanford University were also developing a similar process, although this one involved the passive transfer of proteins.

Burnette knew of both techniques and believed his to be simpler and more universal. However, the reviewers of his paper on its first submission deemed it 'pedestrian'. Despite this rejection, Burnette continued to distribute preprints amongst his colleagues. His method was eventually published in Analytical Biochemistry in 1981. Since then, the western blot has been cited more than 5,000 times. Researchers refer to both Burnette and Towbin when citing western blotting.

About BioTechniques

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