Kinase glo luminescent kinase assay kit

ATP hydrolysis activity was evaluated using the Kinase Glo Luminescent Kinase assay kit (Promega). Beads prepared as described above were distributed in increasing quantities in white 96-well plates in washing buffer (20 mM Tris pH 7.5, 1 mM DTT, 200 µM activated sodium orthovanadate, Roche cOmplete EDTA-free anti-protease cocktail) to 20 µL per well. ATP buffer (washing buffer containing 3.3 µM ATP, 16.7 µg/mL MBP and 3.3 mM MgCl₂) was then added to a final volume of 50 µL per well. JNK1 was used as a kinase positive control. After incubation at 37 °C for 2 h shaking at 54 rpm in the dark, Glo reagent equilibrated to room temperature was added (50 µL per well) and the plate was incubated shaking at 100 rpm for 10 min in the dark at room temperature before measuring the luminescence on a TECAN Spark 10 M luminometer (see Supplementary Table S4 for raw data).

Kinase-Glo® luminescent kinase assay was used to screen compounds for activity against CDC7. Human recombinant full-length CDC7/DBF4 kinase, substrate PDKtide (KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC) and reaction buffer was purchase from Promega (CDC7/DBF4 Kinase Enzyme System, Cat#: V5088, Promega Biotech Ibérica, SL). The enzymatic reaction was performed in a total volume of 40 µL in white 96-well plates. In a typical assay, 10 µL of test compound at a final concentration of 10 µM, 10 µL of enzyme solution (25 ng/well), 10 µL of ATP solution (1 µM/well) and 10 µL of PDKtide substrate (0.2 µg/µL/well) were added to each well. The final DMSO concentration in the reaction mixture did not exceed 1%. After 60-min shake-incubation period, the enzymatic reaction was stopped with 40 µL of Kinase-Glo reagent (Kinase-Glo® Luminescent Kinase Assay kit, Cat#: V6711, Promega Biotech Ibérica, SL). Glow-type luminescence was recorded after 10 min using a GloMax® Discover Microplate Reader (Promega Biotech Ibérica, SL). The activity is proportional to the difference of the total and consumed ATP. The inhibitory activities were calculated based on maximal activities measured in the absence of inhibitor.

This assay is more accurate for measuring higher protein kinase activities as it directly measures the consumption of co-factor ATP during the phospho-transfer. Kinase-Glo^® Luminescent Kinase Assay kit was purchased from Promega. The primary reaction consisted of the kinase, substrate and ATP in 60 mM HEPES, pH 7.5, 3 mM MgCl₂, 3 mM MnCl₂, 3 μM Na₂VO₄, 1.2 mM DTT and 0.05 mg/ml BSA. After 1 h of incubation at 30°C, 20 μL of Kinase-Glo^® Reagent was added and mixed at room temperature for 10 min, and luminescence was measured on a GloMax^® 96 microplate luminometer (Promega). All the kinases used were purchased from ProQinase. For GSK3β we used 140 ng of kinase, 1 μg of GSM (Millipore), or 1 μg of SRPK1, and 1 μM ATP. For each kinase reaction, control reactions were made without substrate or without kinase. 500 μM ibuprofen (Sigma-Aldrich) was added to the reactions, using DMSO as control. As specific inhibitor for GSK3β we used 10 μM CHIR99021 (BioVision). Note that all the above conditions included also 25 μM SRPIN340 (kindly provided by Dr. Masatoshi Hagiwara) in order to inhibit SRPK1 kinase activity.

The in vitro ATPase activity of UspE was measured by quantifying the amount of ATP remaining in the solution following a kinase reaction using a Kinase-Glo® Luminescent Kinase Assay Kit (Promega, Fitchburg, WI, USA). The assay was performed in a 96-well plate in a kinase reaction volume of 50 μl containing 10 mM MgCl₂,5 μM ATP,10 mM HEPES (pH 8.0) and 150 mM NaCl. The reaction was initiated by adding the protein to a final concentration of 0.4 mg/ml-3.2 mg/ml. The reaction mixture was kept at 310 K for 20 min in a water bath. Reaction mixtures containing no UspE were used as negative controls. The kinase reaction mixture was incubated with 50 μl of ATP detection reagent. The plates were then incubated for another 10 min at 310 K. The Synergy2 Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA) was used to collect the relative light unit (RLU) signal. The luminescent signal was positively correlated with the amount of remaining ATP and inversely correlated with the amount of kinase activity.