Proteon manager

SPR-based measurements
of protein–protein interactions were performed on a Biacore
3000 (GE Healthcare) and a Bio-Rad ProteOn XPR36 instrument, essentially
as described previously.^{20 (link)} Briefly, purified
His₆-EhPAK4 PBD and His₆-EhPAK5 PBD proteins
were separately immobilized on an NTA biosensor chip using covalent
capture coupling.^{31 (link)} EhRacC, EhRacC^Q65L, EhRacD, EhRacG, or EhRho1 was injected in 30–100
μL volumes at increasing concentrations. Experiments were performed
in a running buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl,
0.05% NP-40 alternative (Calbiochem), 50 μM EDTA, and 1 mM MgCl₂. Background changes in refractive index upon injection of
samples were subtracted from all curves using BIAevaluation version
3.0 (GE Healthcare) or ProteOn Manager (Bio-Rad). Equilibrium binding
analyses were conducted as previously described^{32 (link)} using GraphPad Prism version 5.0 to determine binding affinities.
Kinetic analyses were performed on triplicate Rho GTPase injections
as previously described.^{33 (link)}