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Loading controls are important tools in Western blot analysis that help researchers to ensure that their samples were loaded equally across the gel. When selecting a loading control, it is important to choose a protein that has a different molecular weight than the protein being studied; this ensures that distinct bands are achieved. Novus Biologicals provides an extensive line of loading controls, including Actin, GAPDH, COX IV, Profilin, Tubulin and VDAC1 antibodies, which are used routinely as loading controls in immunoassays.
Beta Actin is one of six Actin isoforms and is highly involved in all cellular functions. Because it is widely distributed throughout all eukaryotic cells, Beta Actin is commonly used as a loading control. One of Novus Biologicals' most popular Beta Actin antibodies is a mouse monoclonal antibody made to a synthetic peptide conjugated to KLH, corresponding to amino acids 1-14 of Xenopus laevis Actin (beta). (NB600-501).
GAPDH, an approximately 38kDa protein, is constitutively expressed in almost all tissues at high levels and is thus a commonly used loading control. Because this protein is expressed in large amounts and is required at all times for an important housekeeping functions, levels of GAPDH mRNA are often used as standards in studies of mRNA expression. Novus' rabbit polyclonal anti-GAPDH antibody (NB300-322) is widely used by researchers as a loading control.
COX IV is expressed at a constantly high level; therefore, it makes an effective loading control in protein assays, with COX IV antibody as the detector. However, some caution is required as COX IV expression is variable. In addition, many proteins run at the same size as COX IV (16kDa), therefore care should be demonstrated when using this protein as a loading control. For these reasons, COX IV antibody is often used with tagged COX IV. For example, Novus' mouse monoclonal anti-COX IV antibody (H00001327-M08) is tagged with GST in order to increase the molecular weight of the protein and thus improve its functionality as a loading control. Novus also has a rabbit polyclonal anti-COXIV antibody (NB110-39115).
Profilin is an ubiquitous, 12-15kDa phosphoinositides and poly-L-proline binding protein that plays a role both in signal transduction pathways and actin filament dynamics. Profilin 1 appears to be highly expressed in most tissues, except for skeletal muscle, whereas Profilin 2 is predominantly expressed in brain and at lower levels also in skeletal muscle, uterus and kidney. Due to their high expression levels in most tissues, Profilin antibodies are often times employed as loading controls.
Tubulin is a popular marker in protein assays, as it is a major component of microtubules and is found in practically all eukaryotic cells. Tubulin is a globular protein which consists of an alpha and beta subunits, which are each 55kDa. Novus Biologicals' rat monoclonal anti-Tubulin antibody (NB600-506) is popularly employed as a loading control in Western blotting.
VDAC1 is an outer membrane mitochondrial protein. It is expressed at steady levels in mitochondria and thus makes a good mitochondrial loading control.
In addition to these frequently used loading controls, additional loading control antibodies can be found at www.novusbio.com/research-areas/loading-controls.html.
For more information on loading controls or to inquire about product collaborations, please contact the Novus Product Development Team by calling 303-730-1950 or via e-mail at firstname.lastname@example.org.