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Because the GAPDH gene is often stably and constitutively expressed at high levels in most tissues and cells, it is considered a housekeeping gene. For this reason, GAPDH is commonly used by biological researchers as a loading control for western blot and as a control for qPCR.
Housekeeping genes and proteins, including β-Actin, GAPDH, HPRT1, and RPLP1, are often used as internal controls in western blots because they are thought to be expressed constitutively, at the same levels, across experiments.
The western blot is routinely used for verification of protein production after cloning. It is also used in medical diagnostics, e.g., in the HIV test or BSE-Test. [9] The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample.
A loading control is a protein used as a control in a Western blotting experiment. Typically, loading controls are proteins with high and ubiquitous expression, such as beta-actin or GADPH. They are used to make sure that the protein has been loaded equally across all wells. [1] [2]
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 ...
One of the most commonly used methods is to perform a Western blot against the protein of interest. [130] This gives information on the size of the protein in addition to its identity. A sample (often cellular lysate) is separated on a polyacrylamide gel, transferred to a membrane and then probed with an antibody to the protein of interest.
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These can be transferred onto a nitrocellulose or PVDF membrane to be probed with antibodies and corresponding markers, such as in a western blot. Typically resolving gels are made in 6%, 8%, 10%, 12% or 15%. Stacking gel (5%) is poured on top of the resolving gel and a gel comb (which forms the wells and defines the lanes where proteins ...