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Loading Controls

Loading control proteins are known to be constitutively and stably expressed at high levels in almost all tissues and cells. Housekeeping proteins, such as GAPDH , beta-tubulin and beta-actin , represent some of the popular loading control proteins. Loading control antibodies help determine whether samples have been loaded equally in each lane. This information is needed to determine whether the difference in levels of the protein of interest in different samples is due to loading variance or sample-to-sample expression level differences.. View more about loading control FAQs.

Loading Control Selection by Your Sample Type, Use the Figure Below

Featured Loading Control

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  • Immunogen: Full length human recombinant protein of human GAPDH (NP_002037) produced in HEK293T cell.
  • Reactivity: Human, Mouse, Rat, Dog, Monkey
  • Application: WB
  • Predicated MW: 35.9kDa
  • Citations:

Why Choose OriGene Loading Controls

a. OriGene offers a wide selection of loading control antibodies for GAPDH, beta-tubulin and beta-actin and HSP90AB for several species.
b. Most are monoclonal antibodies provided under rigorous quality control on a panel of cell lines.
c. HRP- and biotin-conjugated versions of these antibodies are also available.

OriGene Loading Control Citations

Loading Control Background

Beta-actin is an isform of actin proteins. Actins are highly conserved proteins that are involved in cell motility, structure, and integrity. There are six different but highly conserved actin isoforms in vertebrates. Four of these isoforms are expressed primarily in striated and smooth muscle cells, whereas the two cytoplasmic β-actin and γ-actin isoforms are ubiquitously expressed. Conserved from birds to mammals, β-actin and γ-actin differ at only four biochemically similar amino acid residues. These amino acid differences confer unique biochemical properties between the two isoforms. Beta-actin is a major constituent of the contractile apparatus.

Beta-actin, also known as a "housekeeping" protein, is usually used as a loading control, for among others, the integrity of cells, protein degradation, in PCR and Western blotting. Loading controls are essential for proper interpretation of western blots. They can be used to normalize the levels of protein detected by confirming that protein loading is the same across the gel.

Beta-tubulin , is a subunit of tubulin. Tubulin is one of several members of a small family of globular proteins. It is the major constituent of microtubules. There are two most common members of the tubulin family: alpha-tubulin and beta-tubulin, and together their dimers form microtubules. The dimers of alpha- and beta-tubulin bind to GTP and assemble onto the (+) ends of microtubules while in the GTP-bound state. After the dimer is incorporated into the microtubule, the molecule of GTP bound to the beta -tubulin subunit eventually hydrolyzes into GDP through inter-dimer contacts along the microtubule protofilament. Beta-tubulin faces the plus end of the microtubule while alpha-tubulin faces the minus end. Dimers bound to GTP tend to assemble into microtubules, while dimers bound to GDP tend to fall apart.

GAPDH, less commonly as G3PDH, is the abbreviation of Glyceraldehyde 3-phosphate dehydrogenase. It is an enzyme of ~37kDa that catalyzes the sixth step of glycolysis and thus serves to break down glucose for energy and carbon molecules. GAPDH is considered a housekeeping gene because this gene is often stably and constitutively expressed at high levels in most tissues and cells. So GAPDH is commonly used by biological researchers as a loading control for western blot and as a control for qPCR. .

Loading controls are usually proteins that exhibit high-level, constitutive expression in the cell type or sample you are examining. This ensures constant expression levels. Thus "housekeeping genes" are frequently chosen for use as loading controls. It is also important that the protein chosen as a loading control has a different molecular weight with the protein of interest so that the bands are distinct and expression levels quantifiable. Popular loading control detection antibodies include anti-β-Actin monoclonal or polyclonal antibodies, anti-COX-4, anti-GAPDH, anti-Tubulin and anti-VDAC/Porin antibodies.

Loading Control References

1. "Entrez Gene: ACTB actin, beta"

2. Lecine P et al. (August 2000). "Hematopoietic-specific beta 1 tubulin participates in a pathway of platelet biogenesis dependent on the transcription factor NF-E2". Blood 96 (4): 1366–1373. PMID: 10942379

3. Barber RD, Harmer DW, Coleman RA, Clark BJ (May 2005). "GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues". Physiol. Genomics 21 (3): 389–95. PMID:15769908

 

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