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. Author manuscript; available in PMC: 2016 Jun 1.
Published in final edited form as: J Biomol Screen. 2014 Dec 22;20(5):655–662. doi: 10.1177/1087057114564057

Figure 2. Assay development to determine kinase concentration, ATP and substrate Km, and DMSO sensitivity.

Figure 2

(A) Progression curves for PIP5K1C reactions with varying concentrations of ATP. (B) Michaelis-Menten analysis of initial rates in (A) to determine ATP Km for PIP5K1C. (C) Progression curves with varying concentrations of PIP5K1C to determine the optimal kinase concentration to obtain ~30% conversion after 40-minute incubation. (D) Michaelis-Menten analysis of progression curves for PI5K1C reactions with varying concentrations of substrate to determine substrate Km (E) Percent conversion of PIP5K1C reactions at various DMSO concentrations after 40-minute incubation to determine DMSO sensitivity of the reaction. (F) Final progression curve for reactions containing all optimized parameters. Mean ± SEM. n=12 reactions.