ELISA, Immunohistochemistry, Immunoprecipitation, Western Blot. Anti-EGFR antibody is specifically designed for ELISA, immunoblotting and immunoprecipitation. Reactivity in other assays is likely, but has not been determined. Recognition of EGFR is independent of the phosphorylation status at tyrosine 1173. No reaction is observed against ErbB-2, ErbB-3 or ErbB-4. 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.
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Description
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.
Immunogen
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.