Due to the considerable scientific and commercial interest in poly(ethylene glycol) (PEG) grafted solid surfaces for biomedical applications, it is important to characterize the properties and behaviour of these modified surfaces. In this study, we applied a unique method to predict the magnitude of interactive forces exerted between a globular protein and PEG-grafted matrices. Carboxyl functionalized solid matrices were covalently grafted with PEG-amine derivatives at varying PEG surface concentrations and layer thicknesses by controlling the grafting reaction parameters. X-ray photoelectron spectroscopy was used to characterize these PEG-grafted matrices using the standard overlay model. A detailed and systematic correlation between PEG layer thickness and the distance between the grafted PEG chains resulted in valuable information regarding protein interactions with these PEG-grafted matrices. Finally, this predicted interaction behaviour was validated with fluorescent images obtained from fluorescein isothiocyanate-labelled Cytochrome c-conjugated solid matrices using confocal laser scanning microscopy. (C) 2010 Elsevier B. V. All rights reserved.