Figure 2

Binding of Dl:Fc to a Notch protein including EGF repeats 11–12. A. Dl:Fc binding to adsorbed N11-20, measured using HRP-conjugated anti-human IgG. In these and all subsequent experiments, error bars are standard deviations of parallel replicate experiments. Note that binding is saturable and therefore likely to be specific. Saturation binding and "Kd" (half-maximal binding) were estimated by the Prizm software package. B. Human IgG bound negligibly to N11-20, indicating that Dl sequences were responsible for most binding seen in panel A. C. Dl:Fc did not bind adsorbed BSA, so most of the binding in panel A was to adsorbed N11-20:V5:His. In subsequent figures, non-specific BSA binding by Fc fusion proteins has been measured and subtracted, so that binding specific to adsorbed N proteins is presented. D. Dl:Fc did not bind specifically to N21-30, another EGF repeat protein of equal length to N11-20, but lacking the known ligand binding site. Note that the small amount of signal detected is mostly present in the absence of added Dl:Fc protein, is not saturable, and is therefore likely to be non-specific.