Journal of Clinical Chemistry and Laboratory Medicine
Open Access

Analysis of Proteins by Western Blotting Method

Maroua Surette^{* *}Correspondence: Maroua Surette, Department of Chemistry and Biochemistry, University Moncton, New Brunswick, Canada, Email:

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Introduction

Western Blot (WB) is a broadly used antibody based technique to detect levels of protein expression in a cell or tissue extract. This method estimates protein levels in a biological sample through antibody binding to a particular protein of interest. The name western blot was given to the technique by W. Neal Burnette. The name western blot was given to the technique by W. Neal Burnette. To get linear signals with most of western blots, loading smaller amounts of protein sample should be somewhere in the range of 1 μg and 10 μg per well. To stay away from under or overloading samples, protein concentration of each sample prior to electrophoresis with a compatible protein assay should be decided. The western blot (some of the time called as protein immunoblot), or western blotting, is a broadly used analytical procedure in molecular biology and immune genetics to detect specific proteins in a sample of tissue homogenate or extract. Western blot method utilizes three components to accomplish its task of isolating a particular protein from a complex: partition by size, transfer of protein to a solid support, and marking target protein using a primary and secondary antibody to visualize. An engineered or animal-derived antibody (known as the primary antibody) is made recognizes and binds to a specific target protein. The electrophoresis film is washed in a solution containing the primary antibody, before excess antibody is washed off. A secondary antibody is added which perceives and ties to the primary antibody. The secondary antibody is viewed through different techniques like staining, immunofluorescence, and radioactivity, permitting aberrant identification of the particular objective protein. The western blot is widely utilized in organic chemistry for the qualitative discovery of single proteins and protein-changes (like post-translational modifications). Something like 8%-9% of all protein-related modifications are assessed to apply western blots. It is utilized as an overall strategy to recognize the presence of a particular single protein inside a complex mixture of proteins. A semi-quantitative estimation of a protein can be gotten from the size and color intensity of a protein band on the blot membrane. Furthermore, applying a dilution series of a filtered protein of known concentrations can be utilized to permit a more exact protein concentration.

Description

The western blot is regularly verification of protein production after cloning. It is also utilized in clinical diagnostics, e.g. in the HIV test or BSE-test. The western blot technique is made out of a gel electrophoresis to isolate native proteins by three dimensional design or denatured proteins by the length of the polypeptide, followed by an electrophoretic transfer onto a membrane and an immunostaining procedure to visualize a certain protein on the blot membrane. SDS-PAGE is by and large utilized for the denaturing electrophoretic separation of proteins. SDS is generally used as buffer to give all proteins present a uniform negative charge, since proteins can be positively, negatively, or neutrally charged. This type of electrophoresis is known as SDS-PAGE (SDS-polyacrylamide gel electrophoresis). Before electrophoresis, protein samples are often boiled to denature the proteins present. This ensures that proteins are separated based on size and prevents proteases (enzymes that break down proteins) from degrading samples. It is a generally used technique for detection of a particular protein in a complex matrix, for example, cell or tissue lysate (for example protein removes). The Western blot analysis shows the exchange of proteins and their detection with antibody probe.

Applications of WB

Protein-DNA interactions: Attachment of DNA binding proteins to specific DNA arrangements is vital to the process of transcriptional regulation. South-western blotting (like western blotching aside from the film is tested with DNA) can be utilized to recognize transcription factors in gene regulation studies.

Protein-protein interactions: These are important for the essential cellular processes. Detection of protein–protein interactions, utilizing a variety of western blotting known as farwestern blotting can assist with recognizing cellular functions and dysfunctions.

Post-Translational modifications (PTMs): PTM affect the protein folding and consequently its function, growing proteome variety. In any case, PTM’s have been related with illness. Consequently, their study is of great interest to researchers and western blot provides one such tool with which researchers can do these types of studies.

Protein isoform detection: Proteins might be expressed in differing isoforms in different cellular states with varying activities or targets. On the other hand, they might require cleavage to become activated.

Epitope mapping: Understanding how and where antibodies bind their target protein will be important for research, diagnostic and therapeutic purposes. There are many apparatuses utilized towards this objective and due to its specificity, western blotting is considered as one of those apparatus.

Subcellular protein localization: Performing western blot examination of various cell divisions permits the area of target proteins in the cell to be determined. Single-cell western blotting has offered great insights in this field and overcomes some of the antibody cross-reactivity issues experienced by other singlecell assays.

Conclusion

Western blot has been utilized to confirm cases of diseases like Human Immunodeficiency Virus (HIV), lyme disease, Bovine Spongiform Encephalopathy (BSE) and aspergillosis. Be that as it may, it is tedious compared with strategies, for example, PCR has been replaced by alternative assays in a number of case including HIV testing. Western blotting is perhaps one of the most used techniques in molecular biology and proteomics.