Western Blot

General: Sometimes loosely termed immunoblot, the Western blot is used to separate/identify protein following gel electrophoresis. This is distinct from Southern blot (DNA) and Northern blot (RNA). Following electrophoresis, a membrane (usually nitrocellulose or polyvinylidene fluoride (PVDF)) is placed upon the gel to blot protein onto it. This process may be as simple as capillary action or with the help of an electric current. The separated proteins are subsequently highlighted on the membrane via labeled antibody.

Limitations/common problems:

• Subjectivity of barely perceptible bands
• Subjectivity of band location, especially in the setting of numerous complex bands
• Small errors in pH or other factors can invalidate an entire run
• Cross-contamination
• Relatively time intensive, with several manual steps

Clinical/common useage:

• HIV confirmatory testing, to highlight multiple antibody patterns. Diagnosis depends on a minimum set of antibodies being present in detectable amounts.

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