Western Blot: Analyzing Protein Extracts via Electrophoresis - Essay Sample

Introduction

The Western Blot Technique is an analytical technique widely used in immunogenetics and microbiology to identify particular proteins from a protein extract. In this strategy, molecular weight and by type through electrophoresis form the basis of the separation of the mixture. The outcomes are then moved to the membrane, which produces a band for each protein incubated with label antibodies. In brief, the sample is transitioned through protein denaturation and protein electrophoresis. A synthetic antibody is then created to bind a particular target protein with a solution containing the primary antibody washing the electrophoresis membrane before the excess antibody is washed off. A secondary antibody is then added to identify and bind the primary antibody. The antibody is screened through some strategies such as radioactivity, immunofluorescence, and staining, which allows indirect detection of the targeted protein extract. Other strategies can be employed, such as immunocytochemistry, immunohistochemistry, and quantitative blot, where antibodies are used to identify protein extracts in cells and tissues by either enzyme-linked immune-sorbent or immune-staining.

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To begin with, the first of the Western Blot Technique Gel Electrophoresis, where the protein samples are separated. The separation may be by electric charge, molecular weight, or isoelectric point. Consequently, the type of separation hugely depends on the nature of the gel and treatment of the sample. However, gel electrophoresis employs straps loaded with sodium dodecyl sulfate and polyacrylamide to control the polypeptides in the denatured state. Additionally, acrylamide concentration determines the resolution of the gel, which implies the less the concentration of the acrylamide, the worse the resolution of lower molecular weight. When voltage is applied in the gel with a negative, they will be denatured by the heat and move towards the positive electrode. These gels are often made by pouring them between two plastic plates or glasses using the protocol section. A marker and the sample will then be loaded on the wells with the gel connected to the power supply. Controlling the voltage is highly important since high voltage can overheat and dismantle the bands.

The second step involves blotting, whereby the mixture is transferred to the membrane after separation. The transfer employs an electric field that stands perpendicular to the gel's surface, making the proteins move out of the membrane and the gel. The membrane is inserted between the positive electrode and the gel surface. The layer consists of the filter papers to protect the gel and a fiber pad at each end. The membrane stands in this position so that the negatively charged proteins can move from the gel to the membrane (Butler, Paul, Chan, Smith & Tolosa, 2019). The transfer is generally regarded as the electrophoretic transfer, and it is preferably done in wet conditions since it is less likely to dry out the gel. The membrane is also important since it provides enough support for the whole process.

Incubation follows shortly after as a vital step in Western Blotting. The process prevents antibodies from sticking on the membrane. In most cases, blocking is done with nonfat dried milk or 5% of BSA diluted in tris-buffered saline. Nonfat dried milk is preferred to other solutions because it is generally cheaper and widely available. According to WalentowiczSadlecka, Dziobek, Grabiec, Sadlecki, Walentowicz, Mak, and DutschWicherek (2019), care should be taken when chosing the blocking solution since milk proteins are not compatible with the detection labels (WalentowiczSadlecka, Dziobek, Grabiec, Sadlecki, Walentowicz, Mak and DutschWicherek, 2019). The primary antibody is often incubated with BSA because it is required in large amounts than the secondary antibodies. The solution also allows the reuse of the antibody in case the blot does not provide a good result. The labeled antibody through enzymes such as horseradish peroxidase (HRP) detects the membrane by the produced signal that corresponds to the position of the target protein. The next step involves quantification, which entails putting into account the data produced since the process is often considered semi-quantitative.

Additionally, even though the process is simple, challenges can arise, which may lead to unexpected results. These challenges often range from uneven spots in the blot, high background on the blot, weak signal or faint bands, no bands totally, and unexpected bands. For instance, unexpected bands can be a result of the degradation of the protease. Gibbons (2014) suggest that if such a scenario occurs, it is advisable to use a fresh sample or distort the antibody. Likewise, blurry bands are a result of air bubbles or high voltage during transfer (Gibbons, 2014). In such a situation, you should make sure the transfer sandwich is appropriately prepared, and the voltage is running at a low voltage.

The Western blot technique is broadly used in biochemistry, especially to identify protein modifications and single proteins. The same ideology is used during HIV test confirmation as the technique detects the HIV antibodies present in the sample. Like the process above, proteins from the HIV infected cells are separated and bolted on a membrane. Additionally, in research by WalentowiczSadlecka, Dziobek, Grabiec, Sadlecki, Walentowicz, Mak, and DutschWicherek (2019), western blot was used to analyze the human leukocyte antigen-G level among individuals with endometrial cancer. The result of the study suggests human leucocyte antigen-G proteins level are higher among women with endometrial cancer. The antigen was also prevalent in other cancerous tissues such as the esophagus, cell lungs, cervix, and ovary. Western Blot Technique is also employed by the World Doping Agency to detect a banned substance that enhances the mass of red blood cells, thus making the body transport more oxygen around the body in the bid to increase performance and stamina.

Conclusion

In summation, the Western Blot Technique is an analytical technique widely used in microbiology to identify particular proteins from a protein extract. The two factors that must be taken into consideration during the separation of these proteins include molecular weight and by type of electrophoresis. The technique involves various processes such as gel electrophoresis, transfer, blocking, incubation, quantification, and evaluation. The Western blot technique is broadly used in biochemistry, especially to identify protein modifications and single proteins with the same concept applied during HIV tests. The technique can also be used to detect the human leukocyte antigen-G level among cancer patients. In a more prevalent application, the Western Blot technique is used by the World Doping Agency to detect banned substances in the blood.

References

Butler, T. A., Paul, J. W., Chan, E. C., Smith, R., & Tolosa, J. M. (2019). Misleading Westerns: Common Quantification Mistakes in Western Blot Densitometry and Proposed Corrective Measures. BioMed research international, 2019.

Gibbons, J. (2014). Western blot: protein transfer overview. North American journal of medical sciences, 6(3), 158.

WalentowiczSadlecka, M., Dziobek, K., Grabiec, M., Sadlecki, P., Walentowicz, P., Mak, P., ... & DutschWicherek, M. (2019). The analysis of human leukocyte antigenG level in patients with endometrial cancer by Western blot technique. American Journal of Reproductive Immunology, 81(1), e13070.