HER3 Antibody

HER3 antibody is ideal for western blotting, ELISA and IP. The human Epidermal Growth Factor Receptor (EGFR) family is a family of receptor tyrosine kinases consisting of 4 members: EGFR, HER2, HER3 and HER4. These receptors are expressed on a wide variety of tissue types and have been implicated in many human cancers, including breast, gastric, colon and prostate. Strong expression of both HER3 and HER2 (also known as p185neu) by tumor cells can be associated with poor outcome for patients. The ligand for HER3 and HER4 is called Neu Differentiation Factor (NDF) or Heregulin. In some instances, antibodies against these receptors prove effective in combination therapy regimens for treatment of the disease. Signaling through the EGFR family involves the combinatorial dimerization of the four known family members and is mediated by at least 8 different hormones, including EGF and the heregulins. Heregulin binding and tyrosine phosphorylation by HER3 or HER4 is enhanced in the presence of HER2. Structurally the members of the EGFR family are 1210 to 1342 amino acids in length with approximately 650 amino acids of the extracellular domain being heavily glycosylated. The extracellular domains show up to 70% sequence homology. Anti-HER3 antibody has been tested by ELISA, immunoprecipitation, and western blot and is specifically designed for immunoprecipitation and can be used for immunoblotting in conjunction with a detection antibody like anti-phosphotyrosine. Reactivity in other assays is likely, but has not been determined. Recognition of HER3 is independent of the phosphorylation status of the molecule. Human prostate cell lines (LNCap) or human breast cell line (MDA-453) are typically used as positive control sources. For immunoprecipitation use approximately 5 µL of the antibody. The immunoprecipitation mix contained the antibody, 25 µL of Protein A-agarose beads and 1.0 ml of lysate (lysate contains approximately 1.0 mg of total protein) . This mixture is rotated overnight at 4°C and then washed 3 times with lysis buffer (used to prepare the lysate) . The resulting bead complex is dissolved in 20-30 µL of 3X SDS-PAGE sample buffer and approximately 15 µL is loaded per lane on an 8% polyacrylamide gel. The combination of immunoprecipitation and immunoblotting was performing using the anti-HER3 antibody for immunoprecipitation followed by immunoblot detection using an anti-phosphotyrosine antibody. The complex is then reacted with HRP Goat-anti-Rabbit IgG and ECL for detection and shows a single HER3 band at 180kDa. Researchers should determine optimal titers for other applications.