Introduction to Western Blotting Technique
Blotting is a technique for the detection of macromolecules. There are three types of blotting methods:-
- Southern blotting – used in the detection of DNA
- Northern blotting – used in the detection of RNA
- Western blotting – used for the detection of proteins.
Western blotting is an analytical technique to study the separated fraction of proteins after electrophoresis.
Western blotting is not only used for the detection minute amount of protein but also used for the detection of a chemical reaction is going on inside the cell as well as the total proteome inside the cell.
It is also very important to understand the protein-protein interaction for different physiological cellular purposes inside the cell.
Western blotting can produce qualitative and semi-quantitative data about the protein of interest. It is an important technique used in cell and molecular biology.
It enables the researchers to identify the specific protein from a mixture of proteins extracted from cells as well as the evaluation of their size and amount.
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Principle of western blotting Technique
Western blotting (protein blotting or immunoblotting) is a rapid and sensitive assay for the detection and characterization of proteins. It is based on the principle of immunochromatography where proteins are separated into polyacrylamide gel according to their molecular weight.
The protein thus separated are then transferred or electrotransferred onto nitrocellulose membrane and are detected using a specific primary antibody and secondary enzyme-labeled antibody and substrate.
The procedure used in western blotting
The procedure of Western blotting includes the following steps:-
1.) Preparation of tissue
we take the samples from the whole tissue or from cell culture. The source of proteins can be bacteria, virus or environmental samples.
The detergents, salts, and buffers are used to encourage lysis of cells and solubilize proteins. Tissue preparation can also be done in cold temperatures to avoid protein denaturing.
2.) Gel electrophoresis
The proteins of the sample are separated using gel electrophoresis.
3.) Transfer of the proteins fractions
Transfer of protein from the gel to nitrocellulose can be achieved by two methods:-
- In capillary blotting- The gel is placed on a wet pad of buffer soaked filter paper and a sheet of nitrocellulose placed on the gel. Buffer is then drawn through the gel by placing a pad of absorbent filter paper followed by a heavyweight on top of the nitrocellulose sheet. Through the capillary action through the gel separated proteins come on a sheet and bind hydrophobically to nitrocellulose sheet. Only 10% to 20% of each protein in the gel is transferred in this way. It is a slow method carried out overnight.
- Electro blotting- It is a quicker method and more efficient is a method to transfer proteins or nucleic acids onto a membrane by using PVDF or nitrocellulose, after gel electrophoresis. The protein or nucleic acid can then be further analyzed using probes such as specific antibodies, ligands like lectin or stains. A current is passed at right angles to the gel which causes a separated protein to electrophorese out of the gel and come into the nitrocellulose with its transferred protein is referred to as à blot.
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4.) Probing the blot
After the transfer of protein on nitrocellulose sheet proteins can be examined. For that probing is required for blot.
5.) The antibody can be used to detect specific proteins.
The blot is firstly incubated in a dilution of an antiserum blot which is then incubated in a dilution of an antiserum (primary antibody) directed against the protein of interest.
The primary antibody will bind to the blot and you can identify the protein of interest.
6.) Visualization in Interaction
In order to visualize this interaction, the blot is incubated further in a solution of secondary antibody which is directed against the primary antibody.
The secondary antibody is appropriately tagged with the enzyme. This enzyme-labeled secondary antibody when incubated with the enzyme.
Substrate solution, an enzyme converts the substrate into the colorful product which precipitates on the nitrocellulose sheet.
7.) Detection of Protein
The presence of the colored band indicates the position of the protein of interest.
The protein of interest can be identified by comparing the blot with the stained gel of the same sample.
Uses of Western blot
It is the most sensitive and specific test for determining the size and amount of protein present in any material.
The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample.
A western blot is also used as the definitive test for Creutzfeldt – Jakob disease, Lyme disease, Hepatitis B infection, and HSV-2 (Herpes Type 2) infection.