Application of western blotting.

Western or Immunoblotting is a quantitative technique employed in microbiology to detect the presence of protein(s) in a mix of other proteins. The term western blotting is a play on words for another quantitative technique for identifying DNA, Southern blocking (named after the biologist Edwin Southern.

Immunoblotting is a relatively straightforward process that uses a series of steps and biological reagents like antibodies and other equipment. Western blotting is one of the most reliable techniques in cell and molecular biology. From the previous statement, one can easily deduce that it has a broad range of applications. In this blog, you will learn more about this revolutionary process and the application of western blotting.

Steps involved in western blotting.

Western blotting can be regarded as a six-step process; they include;

Sample preparation: This process involves breaking down the protein derived from cells or tissue samples through homogenization, sonication, etc. Buffers and inhibitors are added to the sample to make the protein soluble and to prevent protein degradation.

Gel electrophoresis: electrophoresis involves using an electric field, a gel (such as polyacrylamide gel), and sodium dodecyl sulfate buffer to separate the proteins based on their molecular weight and isoelectric point.

Protein transfer: This step involves the transfer of proteins onto a membrane before they are incubated.

Blocking: Blocking treats the membrane with non-fat dry milk or bovine serum albumin to prevent non-specific binding of the antibodies to the membrane.

Incubation: Incubation is a two-step process that employs antibodies that selectively bind to the protein of interest. The primary antibody in western blotting first binds to the protein; then, secondary antibodies with radioactive fluorescent or luminescent markers bind to the primary antibody.

Detection and visualization: The final stage of the blotting process identifies the bound secondary antibody (indirectly highlighting the protein of interest) for chemiluminescent, fluorescent, radioactive, or colorimetric detection.

Applications of western immunoblotting.

Qualitative analysis of proteins.

Western blotting is one of the most effective ways of identifying and visualizing proteins and ascertaining the extent of post-translational modifications. Blot stripping can identify multiple proteins in one membrane. It is estimated that nearly 10% of protein-related publications use western blotting. This makes it vital for microbiological and medical fields such as cancer research, disease control, and prevention, biotechnology, food biology, etc.

Disease testing.

Scientists use blotting to test markers of diseases for various diseases. For example, immunoblotting is one of the methods used to determine the presence of HIV in a human sample. The process doesn’t recognize the virus, but it identifies HIV antibodies found in a human serum sample. Other notable areas where blotting is employed in testing include the diagnosis of tularemia, Creutzfeldt-Jakob Disease, Lyme disease, etc.

Doping regulations.

You may be surprised to learn that this qualitative technique has relevance in competitive sports. On many occasions, athletes use under-handed tactics to gain an unfair advantage during competitions. One such tactic includes blood doping. Blood doping refers to the use of substances and other techniques to enhance an athlete’s performance, particularly the ability to transport oxygen. This could be done by transfusion, the use of erythropoietin (EPO), and synthetic oxygen carriers. Thankfully, erythropoietin, a protein, is easily detectable by blotting.

Western blotting was crucial during the anti-doping campaign of the 2014 FIFA world cup.

Advancement in proteomic technology.

Advancements in technology have provided many solutions to the same problems encountered in western blotting. The many steps required have some impact on the final results. With each step, human error is likely to affect the results.

Repetition of blotting procedures that reprobe the membrane (such as in the case of stripping) leaves more room for error. Other innovations in biochemical engineering like one-step probing can improve the process, reduce errors, increase sensitivity, and lead to more accurate results.

Conclusion

Western blotting is a vital semi-quantative technique employed in cell and molecular biology. It has a wide range of applications, from simply detecting proteins, and testing for the presence of viruses to ensuring doping compliance in athletes.

Although it is widely used, researchers and scientists still face some challenges. It is not an easy task and requires a significant amount of skill. With modern science, some of the steps have been automated that have significantly improved the speed, efficiency, and quality of results.