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. Author manuscript; available in PMC: 2014 Apr 1.
Published in final edited form as: Mol Plant. 2012 Dec 19;6(4):1274–1289. doi: 10.1093/mp/sss158

Figure 3.

Figure 3

In Vitro Interaction Analysis.

(A) Quantitative analysis of the Michaelis–Menten kinetics of the CPK3–TPK1 interaction reveals a KM of 3.9 ± 0.6 μM or 4.9 μM (according to analysis using the Hanes plot (B). The substrate conversion rate vmax is calculated to 2.7 ± 0.2 × 10−3 μM s−1 (for [Etotal] = 0.3 μM) providing a turnover number kcat of 9 × 10−3 s−1.

(C) Binding curve as determined from interaction analysis of CPK3 and TPK1 by Microscale Thermophoresis. Analysis of the concentration-dependent thermophoresis and temperature jump of the CPK3 GST–TPK1 interaction yields an affinity of 30 ± 0.9 μM. The normalized fluorescence is plotted for different concentrations of GST–TPK1 (substrate) in the presence of a constant concentration of CPK3 (35 nM), the latter of which was labeled with the fluorescence dye NT-647 via amino-coupling.

(D) The thermophoretic depletion was measured via the fluorescence of the dye-labeled CPK3 kinase in a spot heated by an IR laser. At time point t = 5 s, the IR laser was switched on, the fluorescence decreased as the temperature increases; at time point t = 20 s, the IR laser was switched off again. The diffusion of the dye-labeled CPK3 (in the presence of varying concentrations of its substrate GST–TPK1) was analyzed, providing the affinity of the kinase–substrate interaction.