Choosing loading control antibodies

Western blotting, or immunoblotting, is a routine method of protein analysis in which specific antigen-antibody interactions allow target proteins to be identified in complex mixtures, for example cell lysates.

The first stage of immunoblotting is to separate the molecules using a technique such as PAGE (polyacrylamide gel electrophoresis). However, to ensure the proteins are correctly loaded, and data is interpreted correctly, it is essential to use loading controls. We at Novus Biologicals have an extensive range of loading control proteins and antibodies in our antibody catalog.

Loading controls are proteins, typically housekeeping proteins, which are expressed at high levels in a constant and constitutive manner (i.e. continuously rather than on demand), in the cell type or sample being studied. They should be of a different molecular weight to the proteins of interest, to ensure distinct bands and quantifiable expression levels are achieved. Loading control antibodies perform several essential functions: they confirm samples have been evenly loaded across the gel; they confirm reagents (buffers etc) are functioning correctly, and they allow the protein assay results to be standardized. By combining Western blot and PAGE electrophoresis with the products on our antibody database, researchers have a powerful array of analytical tools with which to perform in-depth analysis of protein samples.

Western blot can be used to analyze many types of sample, from tissue homogenates and cell lysates, to subcellular components such as nuclei and mitochondria. Antibody suppliers have to accommodate this broad spectrum with appropriate loading controls. Whereas actin, GAPDH and tubulin antibody products are fine for cytoplasmic and whole-cell lysates, more specific reagents are required for those working with individual organelles.

As one of the leading global antibody suppliers, we have addressed this problem. Our antibody database contains clearly catalogued nuclear, cytoplasmic, stem cell and mitochondrial markers. Recently updated are our range of beta-actin, profilin, tubulin, GAPDH and COX-IV loading control antibodies.

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