Anti-EGFR Antibody

EGFR is a transmembrane glycoprotein that is a member of a family of protein tyrosine kinases crucial to maintaining a normal balance in cell growth and development. Growth factor receptors are involved not only in promoting the proliferation of normal cells but also in the aberrant growth of many types of human tumors. For example, the epidermal growth factor receptor (EGFR) is mutated and/or over-expressed in many common solid human squamous cell carcinomas including breast, brain, bladder, lung, gastric, head & neck, esophagus, cervix, vulva, ovary, and endometrium. Over-expression of the EGFR gene occurs in carcinomas with and without gene amplification. EGFR and ErbB-2 are particularly important in breast cancer because increased production or activation has been associated with poor prognosis. EGFR belongs to a family of growth factor receptors, which also includes ErbB-2/HER-2/neu, ErbB-3/HER-3/neu and ErbB-4/HER-4/neu. EGFR can heterodimerize with each of the members of this family.

This whole rabbit serum was prepared by repeated immunizations with a peptide synthesized using conventional technology. The sequence of the epitope maps to a region near the carboxy terminus which is identical in human, mouse and rat EGFR.

Cancer, Growth Factors/Hormones, Oncoproteins, Oncoproteins/Suppressors, Protein Phosphorylation, Receptor Tyrosine Kinases, Signal Transduction, Tumor Biomarkers, Tyrosine Kinases

This antiserum is directed against human epidermal growth factor receptor (EGFR) and is useful in determining its presence in western blotting and immunoprecipitation experiments. This antibody can detect EGFR from human, mouse and rat sources. Reactivity of this antibody with EGFR from other species is unknown. No reaction is observed against ErbB-2, ErbB-3 or ErbB-4.

Store vial at -20°C prior to opening. Aliquot contents and freeze at -20°C or below for extended storage. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4°C as an undiluted liquid. Dilute only prior to immediate use. Expiration date is one (1) year from date of opening.

Anti-EGFR antibody has been tested by and is specifically designed for ELISA, immunoblotting, immunoprecipitation, and immunohistochemistry. Reactivity in other assays is likely, but has not been determined. Recognition of EGFR is independent of the phosphorylation status at tyrosine 1173. A431 cells, keratinocytes in normal epidermis, or placenta are typically used as positive control sources. The antigen is typically localized in the cell membrane. For western blotting, good results are also achieved on PVDF membranes blocked with 5% lowfat milk diluted in TTBS for 1 hour at room temperature. Also, dilute the primary antibody and secondary in 5% lowfat milk in TTBS. Anti-EGFR can be diluted up to 1:10,000 for immunoblot depending on the cell line and the amount of EGFR in a particular lysate. For immunoprecipitation, use approximately 10 µl of the antibody. The immunoprecipitation mix should contain 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 should be 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.