How the Western Blot Test is Done
In a laboratory, the western blot test is done by separating proteins in a sample via gel electrophoresis and then transferring them onto a nitrocellulose or PVDF membrane that tightly binds proteins. Antibodies against a specific protein are added to the membrane, and the bound antibodies are detected using a colorimetric method. A report is generated that is then interpreted, usually with reference to a set of standards. Find out bosterbio.com
For a typical HIV blot, the goal is to see whether antibodies in a sample of blood stick to proteins on the surface of the virus envelope. The exact algorithm used to declare a result positive or indeterminate for HIV testing varies and depends on the combination of antibodies present in the sample, so that the results are consistent with known virologic features.
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Proteins in the sample are separated by size by gel electrophoresis, then transferred from the gel to a nitrocellulose or PVDF membrane. This step is often followed by a block in 5% BSA or nonfat dry milk to reduce background, a procedure that prevents antibodies from binding to the membrane nonspecifically.
The blot is then incubated with secondary antibody to the target protein and a soluble dye that binds to a peroxidase (color-producing enzyme) covalently attached to the secondary antibody. The incubation is stopped by washing away the unbound dye, and the protein level is evaluated on a photographic film or by densitometry. Newer chemiluminescent detection methods can also be used.…