Direct visualization of epidermal growth factor receptors (EGFR) in A431 and placental cell membrane by western blot with 125I-EGF

Using the western blot technique, they have devised a new procedure that allowed the direct visualization of both the 150KD and the 170KD forms of EGFR by its natural ligand, 125I-EGF. A431, and placental plasmalemma were purified and solubilized in either SDS-PAGE buffer (without DTT, EDTA) or Triton X-100 (0.5%), resolved on PAGE and electrophoretically transferred onto nitrocellulose (NC) paper. In the absence of boiling, SDS did not denature the EGFR. Although EGER band can be detected after hybridization with 125I-EGF, the receptor signal was considerably improved with the addition of 0.1% Tween-20. The binding of 125I-EGF to the both the 150KD and the 170KD bands of the EGFR was specific, reversible and increased with the amount of membrane protein present. The direct visualization of the EGFR using its natural ligand eliminated the necessity for the time consuming antibody preparation. Presently, they are using this technique to identify specific receptors for other ligands