Eastern Blot- Definition, Principle, Steps, Results, Applications


Eastern blot is a biochemical technique that is used to analyze protein post-translational modifications (PTM) such as lipids, phosphates, and glycoconjugates . It is most often used to detect carbohydrate epitopes on proteins and lipids . Thus, eastern blot can be considered an extension of the biochemical technique of western blot, which is more popular and widely used .

Eastern blot is an immunoblotting technique that depends on the specificity between the protein of interest and a probe in order to identify the biomolecule of interest in a mixture of different molecules. The proteins are electrophoresed on polyacrylamide gel in order to separate them from the mixture. The proteins are then transferred to a nitrocellulose or nylon membrane, where the target molecules are detected by their specific interaction with the probes. The identification of the interaction can be made either through the use of a radioactive probe or by the use of a secondary tagged molecule like in the case of ELISA.

Eastern blotting, like all other blotting techniques, is based on the antigen-antibody interactions where the specificity and extent of interaction determine the result. Eastern blotting is similar to lectin blotting as both of them are primarily used for the detection of carbohydrate epitopes on proteins and lipids. The use of eastern blotting for the detection of smaller molecular compounds is essential as the detection of these molecules cannot be achieved by immunostaining.

Eastern blot has been slightly modified to form a new form of blotting technique called far-eastern blotting, which is used to study lipids that are separated by chromatography. Far-eastern blotting uses antibodies or lectins to stain lipids transferred to polyvinylidene fluoride membranes.

Eastern blot has many applications in the analysis of post-translational modifications in proteins and the detection of different plant products. It also helps to study the nature of interactions between different molecules by the use of ligands. Eastern blot has been extensively used to compare modifications in proteins obtained from different bacterial species.

However, eastern blot also has some limitations such as its complexity, requirement of large amount of sample, difficulty in quantification, and potential destruction of protein structure.

In this article, we will discuss the principle, steps, results, applications, and limitations of eastern blot in detail.

Explanation of the principle of Eastern Blot

Eastern blotting is a molecular biology technique that allows the detection and identification of post-translational modifications in proteins, such as glycosylation, phosphorylation, acetylation, etc. These modifications can affect the structure, function, and interactions of proteins, and are often involved in various biological processes and diseases.

The principle of eastern blotting is based on the specific binding of a probe to the modified protein of interest. The probe can be a monoclonal antibody (MAb), a lectin, a ligand, or any other molecule that recognizes the modification. The probe can also be labeled with a radioactive or enzymatic tag for detection.

The general steps of eastern blotting are as follows:

  • The proteins are separated by thin layer chromatography (TLC) on a silica gel plate. TLC is a method that separates molecules based on their polarity and size. The proteins are applied to the plate and then eluted with a solvent. The solvent moves along the plate by capillary action and carries the proteins with it. The proteins with different polarity and size will migrate at different rates and form spots on the plate.
  • The proteins are transferred from the TLC plate to a membrane, such as polyvinylidene difluoride (PVDF) or nitrocellulose. This can be done by pressing or heating the plate and the membrane together, or by applying an electric current through them. The transfer ensures that the proteins retain their position and orientation on the membrane.
  • The membrane is treated with sodium periodate (NaIO4), which oxidizes the carbohydrate groups on the proteins and forms aldehyde groups. These aldehyde groups can react with bovine serum albumin (BSA) in alkaline conditions and form hapten-BSA conjugates on the membrane. These conjugates serve as antigens for the MAbs that recognize the modification.
  • The membrane is blocked with a solution of skim milk or another protein to prevent nonspecific binding of the probe. The membrane is then incubated with the probe that binds to the modified protein of interest. The probe can be either directly labeled with a tag or indirectly detected by a secondary antibody that binds to the probe and carries a tag.
  • The membrane is washed to remove any unbound probe or antibody. The tag on the probe or antibody is then visualized by adding a substrate that produces a color or radioactivity. The intensity and location of the signal indicate the presence and amount of the modified protein on the membrane.

Eastern blotting is similar to other blotting techniques, such as western blotting, northern blotting, and southern blotting, in that they all use electrophoresis, transfer, and hybridization steps. However, eastern blotting differs from them in several aspects:

  • Eastern blotting uses TLC instead of gel electrophoresis to separate proteins. TLC has some advantages over gel electrophoresis, such as higher resolution, faster separation, lower cost, and less sample consumption.
  • Eastern blotting detects post-translational modifications in proteins rather than their primary structure or nucleic acid sequence. Therefore, eastern blotting requires probes that are specific for the modification rather than for the protein itself.
  • Eastern blotting can detect modifications in both proteins and lipids, whereas other blotting techniques are mainly used for nucleic acids or proteins.

Eastern blotting is a useful technique for studying post-translational modifications in proteins and their roles in various biological phenomena. However, it also has some limitations, such as complexity, sensitivity, specificity, and quantification issues. These will be discussed in more detail later in this article.