Western blot detection of epidermal growth factor receptor from plasmalemma of culture cells using 125I-labeled epidermal growth factor.
We have developed a novel Western blot procedure for the detection of epidermal growth factor (EGF) receptors within a complex mixture of membrane proteins. Purified cell membranes from either human placenta or cultured A431 cells were solubilized, resolved by electrophoresis, and electroblotted onto nitrocellulose paper. With 5-15% gradient gels, electroblotting was completed in 2 h and both the high- and low-molecular-weight proteins were transferred evenly onto the nitrocellulose, as indicated by the radiolabeled protein markers. Upon hybridization with 125I-EGF, the membrane receptor was identified as two adjoining bands on the nitrocellulose of 150 and 170 kDa. Binding of 125I-EGF to the immobilized membrane receptor was specific and was displaced by excess unlabeled EGF. The receptor signal on the autoradiogram was optimized when 1% hemoglobin and 0.05% Tween 20 were present during the hybridization. The ligand-binding activity of the immobilized receptor was not affected by sodium dodecyl sulfate detergent or ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, but was drastically reduced by either heat denaturation or the addition of dithiothreitol to the membrane samples. Using this method, we were able to demonstrate that no noticeable difference was observed between the pre- and postphosphorylated EGF receptors in their ability to bind to 125I-EGF. Because it allows both identification and purification of a receptor from a mixture of proteins, this protocol should have general application in characterizing various receptor-ligand systems.
Lin
P H
PH
Life Science Division, University of California.
Selinfreund
R
R
Wharton
W
W
D002462:Cell Membrane / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor; D066246:ErbB Receptors / Q000032:analysis*; D006801:Humans; D007158:Immunologic Techniques; D008024:Ligands; D010766:Phosphorylation; D011136:Polysorbates; D011869:Radioligand Assay; D014407:Tumor Cells, Cultured / Q000032:analysis*
D008024:Ligands; D011136:Polysorbates; D004815:Epidermal Growth Factor; D066246:ErbB Receptors