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Sunday, November 28, 2021

The Principle and Procedure of Western Blot

When talking about blotting in science, it is a term that refers to the transfer of biological specimens from a gel state to get a membrane and other subsequent findings that come up on the surface of the membrane. 

What is western blot?

Western blot is a method that is used in analyzing protein. Western blot is also known as immunoblotting. Qualitative and semi-qualitative analysis is used in the analysis of protein through western blot, which is a procedure in cell and molecular biology. In detecting the antigen of the protein through the analysis, the western blot service uses an antibody to get the result. In getting an accurate result using western blot, three elements are used in doing this: protein separation by size, transfer of protein to a solid support, and protein markings through primary and secondary antibodies for visualization. This protein analysis was introduced in 1979 by Towbin et al.

The principle of western blot

The principle of western blot entails the identification of a particular protein from the combinations of other proteins. This principle centers on immunochromatography, which separates proteins into polyacrylamide gel. It is performed using polypropylene gel electrophoresis. After the protein has been separated, it is transferred or electro transferred to a nitrocellulose membrane. This protein is detected through specific primary antibodies and secondary enzyme-labeled antibodies and substrates.

The procedure of western blot

Western Blotting - MyBioSource Learning Center

In carrying out western blot analysis services, different steps are involved in getting accurate results from the protein analysis.

Sample preparation/extraction of protein

Extracting protein can be done through different samples like tissues or cells. In western blot, cell lysate is the most common and used sample. In extracting protein from cells, it is done through chemical or mechanical lysis of the cell. In the process, protease inhibitors are used to prevent denaturation of protein at cold temperatures. Spectroscopy determines the concentration of the protein. After the protein is extracted, it is then diluted in a buffer containing glycerol, which helps in the proper sinking of the sample. In monitoring the movement of the protein, bromothymol blue is added to the sample.

Gel Electrophoresis

The commonly used gel is known as polyacrylamide gel and has buffers loaded in sodium dodecyl sulfate. The proteins are separated through electric charge, isoelectric point, molecular point, or using all the methods. When a voltage is applied, smaller proteins move faster in the sodium dodecyl sulfates (SDS) than large proteins. When protein is negatively charged, it migrate towards the anode pole when a voltage is applied.

Electroblotting / protein transfer

After the protein has been separated through gel electrophoresis, it is then moved to nitrocellulose paper using capillary action. This makes the protein accessible to antibody detection. This electroblotting uses an electric field through the gel, which causes the transfer of protein to the membrane. This can be done in semi-dry or wet conditions. The wet condition is proven to be more reliable than the dry.

Blocking 

In western blotting, blocking is crucial in the procedure. Blocking prevents the antibodies from binding to the membrane. This is because antibodies are also proteins, and if not blocked, they can bind nitrocellulose paper. The blocker that is commonly used is the BSA and non-fat dry milk. This blocking reduces the noise in the final product and gives a clear result.

Antibody incubation 

The primary antibody binds itself to the target protein after blocking and incubating the primary antibody with the membrane. In minimizing background and removing unbound antibodies, phosphoprotein western blot services wash the membrane with an antibody-buffer solution which is very helpful and effective. After the membrane has been rinsed, the membrane is exposed to an enzyme, a conjugated secondary antibody. After the incubation of the secondary antibody, the secondary antibody can bind with the primary antibody, which has been reacted upon by the targeted protein.

Protein detection and visualization

In visualizing the action of the enzyme, the reaction mixture with the specific substrate is incubated. A colored substance is generated when the second enzyme is bound with the secondary antibody. Through this, the densitometry and the location of the targeted protein are known. By comparing the protein bands with the marker, the size approximations are taken. Several detection methods are available, but the most common method is electrochemiluminescence (ECL).

Applications of western blot technique: From bench to bedside - Meftahi -  2021 - Biochemistry and Molecular Biology Education - Wiley Online Library

Conclusion

The western blot principle and procedure is a very specific and sensitive test used to determine the amount of protein present in any material or substance. Western blot procedures are a confirmed technique for testing HIV, and it detects the anti-HIV antibody in the spectrum of patients. Western blot service price is not so expensive if you choose to use it to carry out the protein test analysis to detect defective proteins.

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