Western blotting has evolved from a technique with unknown potential to an essential research protocol in several scientific fields.
2020 marked the 40th anniversary of western blotting (also known as “protein blotting” or “immunoblotting”), an analytical technique that researchers use to detect and quantify specific proteins in complex samples. Although the western blot’s potential was unclear at its inception between 1979 and 1981, researchers have used the technique to determine levels of protein expression and changes in protein size with much success over the past four decades. Researchers now use the western blot in several applications, spanning from molecular biology and diagnostics to biotechnology, proteomics, and immunogenetics.
Here, the life sciences journal BioTechniques celebrates western blotting, examining its history, the steps in the western blot assay workflow, the protein detection methods that researchers can use, and the challenges that can arise from the process.
Western Blotting’s Roots
The biochemist W. Neal Burnette coined “western blotting” after the already established Southern and northern blots and after the location of his lab on the west coast of the U.S. Several journals rejected Burnette’s original paper on the western blot, but he shared the paper with his colleagues, who passed photocopies of the paper through their community. Two years later, a peer-reviewed journal published the paper, and, 40 years later, the western blot has proven its enormous value: It is now a standardized technique in labs around the world.
The Western Blotting Assay Workflow
Researchers follow the following steps to perform a typical western blot.
1. Electrophoresis: Researchers separate the proteins in a sample, usually by size, using gel electrophoresis.
2. Transfer the proteins to a membrane: Researchers transfer the proteins to a blotting membrane, which is usually made of PVDF or nitrocellulose. Researchers probe the membrane with a primary antibody that is specific to the target protein.
3. Apply a blocking buffer: Researchers use a blocking buffer to prevent the primary antibody from binding to the membrane.
4. Incubate with a primary antibody: Researchers apply a primary antibody, which binds to the target protein.
5. Wash the blot: Researchers wash any unbound primary antibody from the membrane.
6. Incubate with a secondary antibody: Researchers apply a secondary antibody that recognizes and binds to the primary antibody. The secondary antibody is conjugated to an enzyme or an alternative material that enables the detection of the target protein, which should appear as a band on the blot.
7. Wash the blot: Researchers wash any unbound secondary antibody from the membrane.
8. Detect the protein: Researchers can select one of various methods to detect the target protein. These methods include colorimetric, luminescent, or fluorescent techniques, and the method should depend on the secondary antibody used.
Western Blotting Detection Methods
Having completed steps 1-7 above, researchers can then choose from a variety of detection methods. Each method requires different equipment and comes with its own pros and cons. These are four of the most common detection methods that researchers use.
1. The Colorimetric Western Blot
Researchers performing colorimetric western blots should use an enzyme-conjugated secondary antibody and a chromogenic substrate for detection. The main benefit of this method is that it doesn’t require any specialist equipment. However, its sensitivity is limited, and researchers can’t strip and re-probe blots to investigate additional targets.
2. The Chemiluminescent Western Blot
Researchers performing chemiluminescent western blots should use an enzyme-conjugated secondary antibody and a luminescent substrate for detection. They can then detect results using either a digital imaging system or an x-ray film and darkroom equipment. The main benefits of this method are that it offers a high level of sensitivity and researchers can strip and re-probe blots. However, results can be non-linear, the signal has a short lifetime, and the equipment needed for detection can be both expensive and difficult to store because of size limitations.
3. The Fluorescent Western Blot
Researchers performing fluorescent western blots should use a secondary antibody conjugated to a fluorophore. Therefore, they don’t need to use a substrate. However, they do need a fluorescence imager to detect results. The main benefit of this method is that researchers can multiplex using different fluorophores. Plus, fluorescent western blots offer better linearity and dynamic range than luminescent or colorimetric methods. However, this method is sometimes less sensitive than chemiluminescence, and, as noted, researchers need access to a dedicated fluorescence imaging system.
4. The ScanLater™ Western Blot Detection System
The ScanLater™ Western Blot Detection System uses a multimode microplate reader platform, which facilitates first-of-its-kind protein detection. The technology uses a secondary antibody that is conjugated to a time-resolved fluorophore and unites the advantages of fluorescent and chemiluminescent methods. The system eliminates the need for a substrate to detect target proteins and offers a signal stability of months (or more), a wide dynamic range, and sensitivity at sub-picogram levels.
Difficulties Associated With Western Blotting
Difficulties sometimes arise from western blotting because researchers must take cells apart by lysis before they can study the components of the cells on a gel. But the lysis destroys cellular structures and disturbs cell integrity. This poses challenges when it comes to understanding processes in intact cells, like mitosis, signal transduction, and cell migration. Furthermore, as researchers perform western blots on a large population of cells, the process loses the heterogeneity between cells.
Poor-quality antibodies can also lead to western blotting challenges because a western blot can only be as effective as the antibodies used. Antibodies are isolated from biological samples, so their quality differs from batch to batch. Fortunately, the availability of good-quality antibodies has improved over recent years, making western blots more effective across the board.
Western Blotting in Modern Research
Western blotting may have started as a new technology with unknown potential. But past decades have seen researchers use western blots to answer a multitude of research questions. Today, protein detection is essential to modern clinical and pharmaceutical research, and western blotting is one of the most frequently employed methods for this research.
The open-access, peer-reviewed journal BioTechniques publishes new insights into lab methodologies each month, examining the reproducibility and effectiveness of these techniques and their associated technologies and tools. Research professionals from disciplines like the life sciences, chemistry, physics, computer science, and plant and agricultural science both read and use the journal to develop their practices. These users can also find additional articles, webinars, infographics, podcasts, and videos on BioTechniques’ multimedia website.