Introduction
Western blot is an invaluable lab technique used to detect proteins in a tissue or blood sample. It helps researchers identify specific protein molecules in a complex mixture of proteins. Since antibodies are used in this technique to mark the target protein, this technique is also known as an immunoblot.
Western Blotting
Principle
In Western blot, gel electrophoresis is used to separate proteins in a sample based on their molecular weight. The separated proteins are then transferred to a solid support, which is then exposed to antibodies that can bind to the target protein. This binding is detected using a chemical or radioactive tag. Large protein molecules need to be denatured before electrophoresis to facilitate their movement in the gel.
Procedure
The technique involves 4 key steps, which are discussed below:
Denaturation of proteins
Denaturing involves unfolding of the protein’s tertiary structure to a linear structure. Detergents such as sodium dodecyl sulfate are commonly used to denature proteins. Detergents also offer a negative charge to the protein molecules, which boosts electrophoretic mobility.
Separation of proteins
The denatured protein sample is loaded onto an electrophoretic gel and an electrical charge is applied. Protein molecules are separated on the basis of their size and electrical charge. Smaller and highly charged molecules move quickly in the gel and go farther than the bulkier molecules.
Transfer to a support membrane
The separated proteins is transferred to a sheet of blotting paper made of nitrocellulose. The pattern of the protein molecules in the gel remains the same in the blotting paper.
Visualizing target protein
A primary or monoclonal antibody is added to the blot, which binds to the target protein. A labeled secondary antibody which binds to the primary antibody is then added, allowing detection of the specific protein.
Applications of Western Blotting
Some of the important applications of Western blot are listed below:
- In the detection of circulating antibodies specific to a single protein or several proteins.
- In clinical diagnosis – in HIV testing to detect anti-HIV antibody in the serum sample or as confirmatory tests for diseases such as epidermolysis bullosa acquisita or paraneoplastic pemphigus
- In the analysis of biomarkers such as hormones, growth factors, and cytokines
- In gene expression studies
Disadvantages
A few limitations of the Western blot technique are listed below:
- Western blot is a very delicate and time-consuming process. A minute imbalance at any level of the procedure can skew the results of the entire process.
- The secondary anti-body can sometimes react with a non-intended protein and this can cause labeling of an incorrect protein.
- Insufficient transfer time can result in the larger proteins not transferring properly. This can cause erroneous bands or no bands at all.
- Well trained technicians are a must for this technique
- Western blot is semi-quantitative at best. Only an approximate estimation and not a precise measurement of molecular weight of the protein is possible
- Primary antibody availability is crucial. If it is not available for a specific protein, western blot cannot be used to detect that protein.
Summary
Western blot is an effective technique for studying protein expression in the lab. By stripping the antibody after immunodetection, identification of even multiple protein targets is possible using this technique. However, newer and more advanced techniques such as immunohistochemistry, flow cytometry, and immunofluorescence are more accurate and sensitive compared to the western blot.
These techniques also allow in situ examination of protein expression. The enzyme-linked immunosorbent assay (ELISA) technique is also replacing western blot in many labs as it is more rapid and simple compared to the complex western blot.
One major advantage is that unfolding of the protein’s 3D structure is not needed for ELISA, which helps in preserving the immunologic epitopes of proteins. However, ELISA cannot detect multiple target proteins, unlike the western blot.
References
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