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. Author manuscript; available in PMC: 2009 Jul 2.
Published in final edited form as: J Biomol Screen. 2008 May 19;13(5):415–423. doi: 10.1177/1087057108318331

FIG. 3.

FIG. 3

Galactokinase (GALK) high-throughput screening (HTS) optimization. (a) Effect of varying amounts of adenosine triphosphate (ATP) and enzyme on GALK activity. GALK reaction was carried out at different ATP concentrations (5, 10, 20 μM) and different GALK amount (0, 0.05, 0.1, 0.15, 0.3 μg) in a final volume of 30 μL. Each condition was carried out in 48 replicates (n = 48). At the end of the reaction time (60 min), 30 μL of Kinase-Glo™ reagent was added. Luminescence was recorded as relative luminescence units (RLU) 10 min thereafter. Absolute luminescence values for different amounts of ATP standards (without GALK and galactose) were also determined using the Kinase-Glo™ reagent, and this information was used to convert the raw RLU to the amount of ATP remaining at the end of the reaction. Each data point represents the mean of 48 replicates. Error bars represent ± 1 SD. The experiment was repeated twice to confirm reproducibility. (b) Z′ factor determination. A 384-well plate was divided into 176 wells each for samples and controls, leaving the 2 center columns (32 wells) empty to prevent crossover of luminescence signals. To the sample wells, we added 0.15 μg GALK in the presence of 5 μM ATP and 3 mM galactose in a final volume of 30 μL. In the control wells, GALK was omitted, but other reaction conditions remained identical. Reaction was carried out at room temperature for 60 min. At the end of the reaction, 30 μL of Kinase-Glo™ reagent was added. Luminescence was recorded as RLU after 10 min. Each data point on the graph represents the luminescence recorded for each well. The mean and the standard deviation were calculated for the samples and controls. Sample/control ± 3 SD were plotted around each data point. The experiment was repeated 3 times to confirm reproducibility. (c) Inhibition of GALK activity by ATP-γ-S. GALK activity was assayed in HTS format in the presence of varying amount of ATP-γ-S (pink squares). To control for potential inhibition of luciferase reaction by ATP-γ-S, a separate GALK assay with GALK omitted was performed (blue diamonds). Error bars represent ± 1 SD (n =32).