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Laboratory Methods

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Western blot

This is an immunoassay test method that detects specific proteins in blood or tissue. It combines an electrophoresis step with a step that transfers (blots) the separated proteins onto a membrane. Western blot is often used as a follow-up test to confirm the presence of an antibody and to help diagnose a condition. Examples of its use include confirmatory HIV and Lyme disease testing.

To perform a western blot test, a sample containing the protein is applied to a spot along one end of a layer of gel. Multiple samples and a control may be placed side by side along one end of the gel in separate "lanes." An electrical current causes the proteins in the sample(s) to move across the gel, separating the proteins by size and shape and forming bands that resemble the steps of a ladder. These sample and control ladders are then "blotted" (transferred) onto a thin membrane that is put in contact with the gel. Labelled or tagged antibodies are then used in a one or two step process to detect the proteins bound to the membrane. For example, to confirm HIV or Lyme antibody tests, the proteins separated are those of the causative organism. A patient’s sample is then added to the blot and any antibodies to the organism are bound and later detected by labeled antibodies to human immunoglobulins. The presence of the certain proteins is interpreted by comparison with known negative or positive control samples in the other lanes.

Khalsa, G. Western blotting. Arizona State University, School of Life Sciences, Mama Ji's Molecular Kitchen [On-line information]. Available online at through

Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. Burtis CA and Ashwood ER, Bruns DE, eds. 4th edition St. Louis: Elsevier Saunders; 2006.

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