c 2 prepare compound stock solutions of 2 5 mm in 100 dmso the stocks are subsequently diluted to 60 u00d7 of the final concentration in 100 dmso and diluted 1 5 in kinase phosphorylation buffer 3 prepare a 75 um working atp solution in kinase phosphorylation buffer add 80 ul to each well for a final atp concentration of 50 um 4 transfer 10 ul of the diluted compounds 0 5 log serial dilutions to each well of the pgt assay plate running each compound in triplicates on the same plate 5 dilute on ice fakcd protein to 1 1000 in kinase phosphorylation buffer dispense 30 ul per well 6 note linearity and the appropriate dilution must be pre determined for each batch of protein the enzyme concentration selected should be such that quantitation of the assay signal will be approximately 0 8 1 0 at od450 and in the linear range of the reaction rate 7 prepare both a no atp control noise and a no compound control signal 8 noise one blank row of wells receives 10 ul of 1 5 diluted compounds in dmso 80 ul of phosphorylation buffer minus atp and 30 ul fakcd solution 9 signal control wells receive 10 ul of 1 5 diluted dmso minus compound in kinase phosphorylation buffer 80 ul of 75 um atp and 30 ul of 1 1000 fakcd enzyme 10 incubate reaction at room temperature for 15 minutes with gentle shaking.