Figure 2: Single-molecule kinetic analysis of the HRas–cRafRBD interaction.
(a) kbind as a function of the cRafRBD concentration. A linear increase in kbind is shown as a comparison. (b) kdiss as a function of the cRafRBD concentration. kblinking from the experiment in Fig. 1j is shown as a comparison. Error bars denote s.e. (n>180). (c–e) Real-time traces of HRas–cRafRBD interaction. Note that the background fluorescence increases with the surface HRas density. We used the same prey concentration of [cRafRBD]=10 nM. (f) Background fluorescence increase as a function of the surface HRas density. Three data sets, obtained with 10 nM (red circles), 20 nM (green triangles) eGFP-cRafRBD and 20 nM recombinant eGFP (blue diamonds), are normalized by the corresponding background fluorescence level measured at a low surface Ras density of 0.38 HRas per μm2. Error bars denote s.d. (n>100). (g) Single-molecule spike frequency as a function of background fluorescence. The data set obtained with 10 nM eGFP-cRafRBD is used. Error bars denote s.e. (n>100).
