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The instrumental limit of detection of our BIAcore 1000 instrument is approximately 10 RU; we consider a monolayer of fibrinogen with ca. 5000 RU approximately complete.
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Fluorescence-based techniques require the attachment of dye molecules to the proteins to enable detection; the conjugated molecular systems of dyes add significant hydrophobic character to the surface of proteins and would probably cause dye-conjugated proteins to exhibit a different adsorption isotherm with a hydrophobic surface than unfunctionalized proteins.
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The data of Mrksich et al. cannot be compared directly with those we report here because they were obtained with SAMs in which the hydrophobic groups were buried below the interface defined by the protruding inert groups, but they are useful in inferring the effects of the size and density of the ligand.
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The analysis that we carried out and the experimental data that we have gathered do not allow us to distinguish between films of adsorbed protein that are continuous or films in which individual protein molecules adsorb in discrete islands. The deviation of the measured values of ΔΦ from the predicted ones (Figure 10), however, is strong indication that the proteins do change shape on the surface.
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