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Western blot workflow. The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. [1]
Normalization of Western blot data is an analytical step that is performed to compare the relative abundance of a specific protein across the lanes of a blot or gel under diverse experimental treatments, or across tissues or developmental stages.
In a generally healthy low-risk population, indeterminate results on western blot occur on the order of 1 in 5,000 patients. [15] However, for those individuals who have had high-risk exposures to individuals where HIV-2 is most prevalent, Western Africa, an inconclusive western blot test may prove infection with HIV-2. [16]
Western blotting allows the detection of specific proteins from extracts made from cells or tissues, before or after any purification steps. Proteins are generally separated by size using gel electrophoresis before being transferred to a synthetic membrane via dry, semi-dry, or wet blotting methods. The membrane can then be probed using ...
The article says: "With confirmatory Western blot, the chance of a false-positive identification in a low-prevalence setting is about 1 in 250 000". This overoptimistic estimation is based on Bayesian probability theory assuming strict independency between the variables tested by Western Blot and those tested by EIA.
The detection of horseradish peroxidase by enzymatic chemiluminescence (ECL) is a common method of detecting antibodies in western blotting. Another example is the enzyme luciferase, this is found in fireflies and naturally produces light from its substrate luciferin.
Similar to a western blot, the far-western blot uses protein–protein interactions to detect the presence of a specific protein immobilized on a blotting matrix. Antibodies are then used to detect the presence of the protein–protein complex, making the Far-Western blot a specific case of the Western blot.
Immunoprecipitation of intact protein complexes (i.e. antigen along with any proteins or ligands that are bound to it) is known as co-immunoprecipitation (Co-IP). Co-IP works by selecting an antibody that targets a known protein that is believed to be a member of a larger complex of proteins.