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Background
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Loading controls are used in immunoblots to normalize the levels of protein detected by confirming that protein loading is the same across the gel. The expression levels of the loading control should not vary between the different sample lanes. Loading controls are usually proteins that exhibit high-level, constitutive expression in the cell type or sample. This ensures constant expression levels. Thus housekeeping genes such as Beta-actin, Alpha-tubulin, and GAPDH are frequently chosen for use as loading controls. Actin is a ubiquitous protein and a major component of the eukaryotic cytoskeleton. Globular tubulin subunits made up of alpha- and beta-tubulin heterodimers are the building blocks of microtubules, one of three types of cytosolic fibers that comprise the cytoskeleton. GAPDH is constitutively expressed at high levels in almost all tissues and cell lines, catalyzes the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate in the presence of inorganic phosphate and nicotinamide adenine dinucleotide (NAD), an important energy-yielding step in carbohydrate metabolism.
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