✍️ Author: Dr Eleni Christoforidou
🕒 Approximate reading time: 4 minutes
Western blotting is an essential molecular biology technique frequently utilised in neuroscience research. This workhorse tool allows for the detection and quantification of specific proteins in a sample, thus aiding in the exploration of complex biological questions.
The process of Western blotting comprises a few key stages:
Gel Electrophoresis: Proteins from the sample are separated according to their molecular weight by an electric field in a gel matrix.
Transfer: The separated proteins are then transferred from the gel to a membrane, typically made of nitrocellulose or polyvinylidene fluoride (PVDF).
Blocking and Probing: To prevent non-specific binding, the membrane is first blocked with a protein-rich solution. It is then probed with antibodies that recognise and bind to the protein of interest.
Detection: A secondary antibody, which recognises the first, is applied. This secondary antibody is linked to an enzyme that catalyses a reaction, producing a detectable signal corresponding to the protein of interest.
Protein Expression Studies: Western blotting can reveal the levels of a specific protein in neuronal cells or tissues, enabling scientists to understand changes in protein expression under different conditions, such as disease states or after drug treatment.
Protein Modification Studies: Western blotting can also detect post-translational modifications of proteins, such as phosphorylation or ubiquitination, which play crucial roles in neuronal function.
Verification of Antibodies: Researchers can use Western blotting to verify the specificity of antibodies they plan to use in other experiments, like immunohistochemistry or immunofluorescence.
For neurodegenerative diseases such as ALS, Western blotting is a vital technique:
Protein Misfolding and Aggregation: Many neurodegenerative diseases involve the misfolding and aggregation of specific proteins. Western blotting can detect these protein aggregates, providing insights into disease mechanisms.
Effect of Therapeutics: If a potential therapeutic is designed to increase or decrease the level of a specific protein, Western blotting can be used to confirm whether the treatment has had the desired effect.
Biomarker Discovery: By comparing protein levels in healthy and diseased tissues, researchers can identify potential biomarkers for disease diagnosis or progression.
Western blotting is a versatile and invaluable tool in neuroscience research, providing critical insights into protein expression and modification. Despite its limitations, such as semi-quantitative nature and labour-intensity, its wide applications, from studying disease mechanisms to validating potential therapeutics, ensure its continuing role in the neuroscience research arsenal.