Binding affinity between full-length Rab5-Q79L and APPL1 (5−419) was quantitatively determined in a surface plasmon resonance (SPR) experiment.
Rab5 was coupled to the SPR biosensor chip in random orientations, and APPL1 (5−419) was applied as the analyte at concentrations of 0.15−12 μM (Supplementary Figure 4).
The dissociation constant, kd, for the Rab5−APPL1 interaction measured from this experiment was 0.9 (±0.7) μM, with kon and koff of 1.3 (±0.6) × 103 M−1 s−1, and 1.2 (±0.4) × 10−3 s−1, respectively.
This kd value is typical for an interaction between a Rab and its effectors (Eathiraj et al, 2005).