A solid-phase assay to evaluate interactions with the GRB-SH2 domain is described. The method is based on the binding of a radio-iodinated 13 amino acid phosphopeptide flanking Y1068 of the EGF receptor to the SH2 domain attached to the surface of a microtiter plate that contains a scintillant as an integral part of the plastic. This proximity-type assay allows binding to be evaluated without washing steps, which significantly increases accuracy over existing methods, since the binding equilibrium remains undisturbed. The IC50 for competition with the unlabeled EGFR-Y1068 peptide was 701 nM and was specific, since peptides known to interact with SH2 domains of P13-kinase or PLC-y were inactive. The new methodology is not only an excellent research tool but, because of its simplicity, it is also ideally suited for high throughput screening.
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