FIGURE 6.
Quantitative analysis of Prp8p/ubiquitin binding by BIA-core surface plasmon resonance. (A) Various concentrations of purified Prp8p fragment were injected over hexahistidine-tagged ubiquitin that was immobilized on a Ni2+-nitrilotriacetic acid (Ni-NTA) sensor chip. Prp8p binding was done at 4°C and was recorded by the instrument as “response units” that were normalized for the ubiquitin density on the chip. (B) Data from A were corrected for background and fit to a binding isotherm (assuming 1:1 stoichiometry) using KaleidaGraph (Synergy) software. The fit indicated an equilibrium dissociation constant (Kd) of 380 ± 70 μM.