Western blot
Introduction
Also known as immunoblotting, the Western blot technique is used to identify specific proteins or antibodies in complex mixtures and has largely overtaken the use of immunoelectrophoresis in research and diagnosis.
Principle
In the Western blot technique, a protein mixture is electrophoretically separated onto a denaturing gel, separating the proteins according to molecular weight. The bands can then be identified by applying enzyme-/radio-labeled antibodies to the mixture and the resulting complexes visualised either by ELISA technique or autoradiography.
Method
- Protein mixture is treated with sodium dodecyl sulfate (SDS), a strong dissociating agent
- Mixture is separated by electrophoresis - takes place in SDS polyacrylamide gel (SDS-PAGE)
- The gel is removed and applied to a protein-binding sheet of nitrocellulose/nylon and an electric current passed through it
- The antigens of interest are detected using enzyme-linked antibodies
- An ELISA reaction is used to detect the position of the antibodies
Applications
- HIV testing- this is the most widely used application of this test, used to determine whether a patient is producing antibodies specific to viral proteins present during an HIV infection
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Originally funded by the RVC Jim Bee Award 2007 |