Eastern Blotting Protocol

In this eastern blotting protocol post we have briefly explained about eastern blotting, principle, components, eastern blotting technique procedure, applications, advantages and limitations of astern blotting technique.

Eastern Blotting Technique

Eastern blotting technique is a molecular biology technique for detecting post-translational modifications in proteins as well as the presence of components such as lipids and carbohydrates. It is an extension of the biochemical technique of western blotting used to detect protein post-translational modification.

Proteins that have been blotted from a two-dimensional SDS-PAGE gel onto a PVDF or nitrocellulose membrane are analysed for post-translational protein modifications using probes specifically designed to detect lipids, carbohydrate, phosphomoieties, or any other protein modification.

Most proteins that are translated from mRNA are modified before they become functional in cells. The modifications are referred to collectively as post-translational modifications (PTMs).

The nascent or folded proteins are then subjected to a battery of specific enzyme-catalysed modifications on the side chains or backbones, if they are stable under physiological conditions.

Principle

Eastern blotting technique works on the same principles as other blotting techniques. It is an immunoblotting technique that uses the specificity of the protein of interest and a probe to identify the biomolecule of interest in a mixture of other molecules.

In the case of eastern blotting technique, the proteins are separated from the mixture by electrophoresis on polyacrylamide gel. After that, the proteins are transferred to a nitrocellulose or nylon membrane, where the target molecules are detected by their specific interaction with the probes.

The interaction can be identified using either a radioactive probe or a secondary tagged molecule, as in the case of ELISA. It is like all other blotting techniques, is based on antigen-antibody interactions, with the specificity and extent of the interaction determining the outcome.

Eastern blotting technique is similar to lectin blotting in that both are used to detect carbohydrate epitopes on proteins and lipids. The use of eastern blotting technique for the detection of smaller molecular compounds is critical because immunostaining cannot detect these molecules.

Requirements

1. Thin Layer Chromatography Plate

2. Transfer Membrane

3. NaIO4 solution

4. Blotting Solution

5. Na2CO3/ NaHCO3

6. Stainless steel plate

Eastern Blotting Protocol

1. After chromatography, the TLC plate is covered with a PVDF membrane to transfer the contents from the plate to the membrane by pressing or heating.

2. Component transfer from the TLC plate to the membrane can also be accomplished by passing an electric current through the structure.

3. The membrane is then treated with sodium periodide to adhere the components to it. It is immersed in the solution for 1 hour before being washed with water.

4. To prepare the hapten-BSA conjugate on the membrane, the membrane is immersed in an alkaline BSA solution (Na2CO3/NaHCO3 buffer).

5. It is blocked with 5% skim milk/PBS for 3 hours before being washed with PBS. MAb, which detects the hapten, was applied to the membrane.

6. The interaction between the MAb and the hapten serves as the foundation for eastern blotting detection.

7. The addition of a second antibody labelled with peroxidase allows the MAb to be detected. Finally, a substrate is added to the membrane, which aids in the staining of the second antibody.

Result

The type of label used in the second antibody influences the outcome of eastern blotting. The appearance of colour or radioactivity indicates a positive outcome. The absence of colour or radioactivity indicates a negative outcome.

Applications

1. The analysis of post-translational modifications in proteins is the most important application of eastern blotting technique.

2. Eastern blotting technique has been used to identify and purify various plant products. Eastern blotting technique can also detect modifications in proteins from various origins.

3. Eastern blotting technique also aids in the investigation of the nature of interactions between different molecules through the use of ligands.

4. Eastern blotting technique has been widely used to compare protein modifications obtained from various bacterial species. Eastern blotting technique is also used to identify carbohydrate epitopes in proteins.

eastern blotting

Limitations

1. Eastern blotting technique is complex, with multiple steps that are prone to error. Eastern blotting technique also necessitates the use of trained personnel.

2. Eastern blotting technique technique necessitates the use of a significant amount of sample, which may be an issue in the case of low-yield productions.

3. Eastern blotting technique makes accurate quantification of proteins and biomolecules difficult.

4. Eastern blotting technique some cases, the interaction between the probe and the biomolecules may result in the destruction of the protein’s tertiary structure.

Further Readings

Reference