Introduction:

Western blotting, also known as protein blotting, is a technique used to analyze and identify specific proteins in a sample. The method is based on the principle of protein separation by electrophoresis and protein detection by antibody binding. It has been widely used in biochemistry, molecular biology, and medical research for the detection and characterization of proteins.

Discovery:

The concept of Western blotting was first proposed by the American biochemist Edwin Southern in 1975. He used a technique called Southern blotting, which is used to analyze DNA, and modified it to analyze proteins. Southern named the technique Western blotting as a reference to his previous work on Southern blotting.

Principal:

The principle of Western blotting is based on the separation of proteins by electrophoresis and detection by antibody binding. Proteins are separated by size and charge in an electrophoresis gel, and then transferred to a membrane, such as nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is then incubated with a specific antibody that binds to the protein of interest, and then a secondary antibody that is conjugated to a detection enzyme is added. The detection enzyme catalyzes a reaction that produces a visible signal, such as a color change or fluorescence.

Procedure (Step-wise):

1. Sample preparation: The protein sample is prepared by extracting it from the source tissue or cell. The sample is then denatured and reduced to ensure that the proteins are in their native state.
2. Electrophoresis: The protein sample is loaded onto an electrophoresis gel, such as polyacrylamide gel, and an electric field is applied. The proteins migrate through the gel based on their size and charge, resulting in a separation of the proteins.
3. Transfer: The proteins are transferred from the gel to a membrane, such as nitrocellulose or PVDF. This process is called electroblotting or western blotting.
4. Blocking: The membrane is blocked with a solution containing a non-specific protein to prevent non-specific binding of the primary antibody.
5. Primary antibody: The membrane is incubated with a primary antibody that binds specifically to the protein of interest.
6. Secondary antibody: The membrane is then incubated with a secondary antibody that is conjugated to a detection enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP).
7. Detection: The detection enzyme catalyzes a reaction that produces a visible signal, such as a color change or fluorescence.
8. Analysis: The band on the membrane that corresponds to the protein of interest is analyzed and the protein is identified.

Applications:

1. Medical research: Western blotting is used to detect and analyze proteins in various diseases such as cancer, HIV, and Alzheimer’s disease.
2. Biotechnology: Western blotting is used to analyze and purify proteins for biotechnology and drug development.
3. Quality control: Western blotting is used to ensure the quality of protein samples used in research and industry.
4. Diagnostics: Western blotting is used in the diagnosis of various diseases such as Lyme disease and West Nile virus.
5. Environmental monitoring: Western blotting is used to detect and analyze proteins in environmental samples such as water and soil.

In conclusion, Western blotting is a powerful technique widely used in biochemistry, molecular biology, and medical research. It enables protein analysis and identification through electrophoresis separation and antibody binding. The method finds applications in disease research, biotechnology, diagnostics, quality control, and environmental monitoring. Western blotting plays a vital role in understanding protein characteristics and advancing various fields of scientific inquiry.