Enzyme concentration curve using proteins as substrates. MBP (0.5 μg/well) was phosphorylated using various amounts of PKCα and IRAK4 (2A) and Histone H1 and PHAS-1 (0.5 μg/well) were phosphorylated using various amounts of PKCα (2B) for 1 hour at room temperature (~25°C) in a 384-well white Optiplate. Following reaction, QTL Sensor was added for 10 minutes at ~25°C. Then, dye-labeled phosphopeptide tracer was added with a final concentration of 0.5 μM for detection of phosphorylation of MBP or 125 nM for Histone H1 and PHAS-1. The plate was incubated for an additional 30 minutes at ~25°C and fluorescence measured.