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How Does Western Blotting Differ from ELISA?

Western Blotting

Two longstanding laboratory techniques that have proven indispensable in proteomics and genetics research, ELISA and western blotting have made largely unheralded but absolutely essential contributions to a number of important scientific and medical discoveries. Each of these trusted immunosorbent assay types has a specific set of applications and offers its own distinct advantages and drawbacks.  Following are the main benefits and limitations of both ELISA and western blotting.

Although it is slightly older than western blotting, ELISA (short for “enzyme linked immunosorbent assay”) remains significantly less time-consuming and technically demanding than its predecessor. Powered by antibody-antigen interactions, the ELISA approach also avoids the precise conditional demands of western blotting, which requires optimal gel concentration and protein isolation among other critical experimental factors.

Despite these drawbacks, western blotting offers a number of distinct advantages when compared to ELISA. Considerably more versatile, western blotting enables researchers to separate proteins according to size, charge, and conformation. This versatility allows scientists to both describe and quantify the targeted protein. Furthermore, while ELISA can only analyze one protein of interest at a time, western blotting enables researchers to detect and characterize several protein targets simultaneously.

In addition to serving as a powerful diagnostic tool in the fields of pathology and general medicine, ELISA has broad spectrum of commercial and industrial quality control applications. The ELISA proceed begins by diluting antigens in a protein mix test sample and then attaching that protein mix to a multiwell plate or another prepared surface. After organizing the sample on this surface according to a pre-determined layout, researchers can add a particular antibody that binds to the antigens of the protein mix. The antibody added to the sample must be linked to a particular enzyme that will drive a protein-detecting reaction during the final stage of the ELISA process. Applying the enzyme’s substrate to the sample, researchers catalyze a natural response that results in a change of color or another clearly identifiable physical marker. The specific characteristics of these markers can enable scientists to detect viral particles and food antigens as well as distinct proteins. 

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An alternative approach to protein detection, western blotting uses a process called gel electrophoresis to separate proteins according to size, charge, or another differential diagnostic factor. Gel electrophoresis involves applying an electric charge to a protein mixture sample through a buffer solution in an electrophoresis chamber with a connected electrode. Researchers then transfer the protein mixture from the gel to a specialized membrane using a wet-transfer “blotting” method. The blotting membrane, which is typically composed of nitrocellulose or PVDF (polyvinylidene difluoride), is subsequently probed with specific marking antibodies that target specific proteins for identification in ways that are quite similar to those in the ELISA process. Since these reagent antibodies are colorless, researchers radioactively label them with fluorescent proteins that tend to be red or green. This coloration allows researchers that use luminescent imaging to measure the size and density of various blotting membrane bands, thereby detecting specific protein properties and characteristics.

Although they differ in significant ways, ELISA and western blotting are similar in their relative importance. Both have led to countless medical innovations that have furthered the effective diagnosis and treatment of a wide range of devastating diseases and disorders. By employing the best immunosorbent assay for the task at hand, researchers will continue to leverage the power of ELISA and western blotting to address challenges that are known as well as those that we have yet to imagine.

 

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