Table of Contents
- Introduction to Eastern Blot
- Principle of Eastern Blot
- Requirements
- Procedure of Eastern Blot
- Result Interpretation
- Applications of Eastern Blot
- Limitations of Eastern Blot
- References
Introduction to Eastern Blot
- Eastern blot is a molecular biology technique used to detect post-translational modifications in proteins as well as the presence of components such as lipids and carbohydrates associated with proteins.
- It was developed as an extension of the western blotting technique, expanding its ability beyond simple protein detection.
- Eastern blot is mainly performed to identify and analyze different biomolecules present in proteins, which helps in comparing post-translational modifications among different species.
- The basic principle and general procedure are similar to other blotting techniques, but the type of molecule or particle being detected varies depending on the purpose of the analysis.
- Eastern blot has been further modified to create a related technique known as far-western blotting, which is specifically used to study lipids that have been separated by chromatography.
Principle of Eastern Blot
The principle of eastern blotting is similar to that of other blotting techniques, following the same basic concept of separation, transfer, and detection.
- It is an immunoblotting technique that relies on the specific interaction between the protein of interest and a probe to identify the biomolecule of interest within a complex mixture.
- In eastern blotting, proteins are first separated by polyacrylamide gel electrophoresis, which allows different proteins to be resolved from a mixture based on their size.
- After separation, the proteins are transferred onto a nitrocellulose or nylon membrane, where the target molecules are detected through their specific binding with probes.
- The interaction between the probe and the target molecule can be identified either by using a radioactive probe or by employing a secondary tagged molecule, similar to the detection system used in ELISA.
- Like other blotting techniques, eastern blotting is based on antigen–antibody interactions, in which the specificity and strength of the interaction determine the accuracy of the result.
- Eastern blotting is closely related to lectin blotting, as both techniques are mainly used for detecting carbohydrate epitopes on proteins and lipids.
- The technique is especially important for detecting small molecular compounds, because such molecules cannot be effectively detected by immunostaining methods.
Requirements
- Thin layer chromatography (TLC) plate is required to separate biomolecules such as lipids, carbohydrates, or modified proteins before transfer.
- Transfer membrane (such as nitrocellulose or nylon) is used to receive and immobilize the separated molecules from the TLC plate.
- NaIO₄ (sodium periodate) solution is needed to oxidize carbohydrate groups, enabling their interaction with detection probes.
- Blotting solution is used to facilitate the transfer of biomolecules from the TLC plate onto the membrane.
- Na₂CO₃ / NaHCO₃ buffer is required to provide the appropriate alkaline conditions during the blotting and detection process.
- Stainless steel plate is used to support the TLC plate and membrane assembly during the transfer process.
Procedure of Eastern Blot
- The TLC plate developed after chromatography is covered with a PVDF membrane to transfer the separated components from the plate to the membrane, either by pressing or heating.
- The transfer of components from the TLC plate to the membrane can also be achieved by passing an electric current through the setup.
- The membrane is then treated with sodium periodate (NaIO₄) to fix the components onto the membrane; it is incubated for 1 hour in the solution and then washed with water.
- The membrane is immersed in BSA solution under alkaline conditions (Na₂CO₃/NaHCO₃ buffer) to form a hapten–BSA conjugate on the membrane.
- The membrane is then blocked with 5% skim milk in PBS for 3 hours and washed with PBS to prevent non-specific binding, after which it is treated with monoclonal antibody (MAb) that detects the hapten.
- The specific interaction between the MAb and the hapten forms the basis of detection in eastern blotting.
- The bound MAb is detected by adding a second antibody labeled with peroxidase.
- Finally, a substrate is added, which allows visualization of the second antibody through staining, revealing the target biomolecules on the membrane.
Result Interpretation
- The result of eastern blotting depends on the type of label attached to the second antibody, such as an enzyme or a radioactive tag.
- A positive result is indicated by the appearance of color (in enzyme-based detection) or radioactivity (in radiolabeled detection).
- A negative result is indicated by the absence of color or radioactivity, showing that the target biomolecule is not present or not detected.
Applications of Eastern Blot
- The most important application of eastern blotting is the analysis of post-translational modifications in proteins, allowing detailed study of how proteins are chemically modified after synthesis.
- The technique has been used to identify and purify various plant products, especially those associated with specific biomolecules.
- Eastern blotting enables the detection of protein modifications from different biological origins, making it useful for comparative biochemical studies.
- It helps in studying the nature of interactions between different molecules through the use of specific ligands that bind to target components.
- The method has been widely applied to compare protein modifications among different bacterial species, supporting research in microbial diversity and function.
- Eastern blotting is also used to detect carbohydrate epitopes on proteins, which is important for understanding glycoprotein structure and function.
Limitations of Eastern Blot
- The technique is complex and involves multiple steps, making it prone to errors and requiring trained personnel for proper execution.
- Eastern blotting requires a relatively large amount of sample, which can be a limitation when working with low-yield or rare biological materials.
- Accurate quantification of proteins and biomolecules is difficult using eastern blotting, reducing its suitability for precise measurements.
- In some cases, the interaction between the probe and the biomolecules can disrupt or destroy the tertiary structure of proteins, potentially affecting the reliability of the results.
References
- Nicholas, M. W., & North, K. N. (2013). North, south, or east? Blotting techniques. Journal of Investigative Dermatology, 133(7), e10. https://doi.org/10.1038/jid.2013.216
- Gershoni, J. M., & Palade, G. E. (1983). Protein blotting: Principles and applications. Analytical Biochemistry, 131(1), 1–15. https://doi.org/10.1016/0003-2697(83)90128-8
- Gershoni, J. M., & Palade, G. E. (1983). Protein blotting: Principles and applications. Analytical Biochemistry, 131(1), 1–15. https://doi.org/10.1016/0003-2697(83)90128-8
- Tanaka, H., Putalun, W., & Shoyama, Y. (2012). Fingerprinting of natural products by eastern blotting using monoclonal antibodies. Chromatography Research International, 2012, Article ID 130732, 1–7. https://doi.org/10.1155/2012/130732
- Taki, T., Gonzalez, T., Goto-Inoue, N., Hayasaka, T., & Setou, M. (2009). TLC blot (far-eastern blot) and its applications. In B. Kurien & R. Scofield (Eds.), Protein Blotting and Detection (Methods in Molecular Biology, Vol. 536). Humana Press. https://doi.org/10.1007/978-1-59745-542-8_55
