Western blot

Western blot workflow

Western Blot results for HIV test

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SDS-PAGE Electrophoresis

Western blot transfer

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Western Blot binding

Western blot (also known as protein immunoblot) is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. The method uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/non-denaturing conditions). The proteins are then transferred to a membrane (typically PVDF or nitrocellulose), where they are stained with antibodies specific to the target protein.

Procedure

The Western blot procedure involves several steps:

1. Sample preparation: The protein samples are prepared by mixing with a SDS-PAGE loading buffer and possibly heating.
2. Gel electrophoresis: The proteins are separated based on their size through SDS-PAGE.
3. Transfer: The proteins are transferred from the gel onto a membrane, making them more accessible for analysis.
4. Blocking: The membrane is incubated with a solution of protein (such as non-fat dry milk) to block nonspecific binding sites.
5. Antibody incubation: The membrane is incubated with a primary antibody specific to the target protein. Then, after washing, it is incubated with a secondary antibody conjugated to a reporter enzyme or dye.
6. Detection: The enzyme or dye is activated, producing a signal that can be detected by various methods, indicating the presence and quantity of the target protein.

Applications

Western blotting is used in both scientific research and medical diagnostics. In research, it helps in identifying specific proteins in a complex mixture and studying protein expression under various conditions. In diagnostics, it is employed for the detection of antibodies in diseases like HIV, Lyme disease, and Hepatitis B.

Advantages and Limitations

The main advantage of Western blotting is its specificity; it can detect a single protein in a mixture of thousands of proteins. However, it is a time-consuming process and requires a significant amount of technical skill to perform accurately. Additionally, quantification can be challenging and less precise compared to other methods like ELISA.

See Also

• ELISA
• Immunohistochemistry
• Northern blot
• Southern blot

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