Kinase glo plus luminescent kinase assay kit

The in vitro preliminary kinase assays human α1β1γ1 AMPK were carried out according to the previous experimental method [33 (link)]. The screened compounds and α1β1γ1 AMPK isoform were provided by Topscience Co. (Shanghai, China) and Huawei Pharmaceutical Co. Ltd. (Shanghai, China), respectively. Generally, each of the evaluated compounds was dissolved in 10% Dimethyl sulphoxide at 10 μM and diluted to a required concentration with buffer solution. Then, 5 μL of the dilution was added to a 30 μL kinase assay buffer and 5 μL AMPK isoform per well. The solution was mixed at 0 °C for 30 min. Next, 5 μL of AMARA petide and 5 μL Adenosine triphosphate (ATP) were added to the well. The enzymatic reactions were conducted at 30 °C for 30 min. The AMPK activity was determined by quantifying the amount of ATP remaining in assay solution with Kinase-Glo Plus luminescent kinase assay kit (Promega, Madison, WI, USA). The luminescent signal is correlated with the amount of ATP present, while inversely correlated with the kinase activity. The mean values from three independent experiments were used for the expression of relative activities. A-769662, a β1-selective AMPK activator reported by Abbott laboratories, was used as a control.

A non-radioactive assay was performed using Promega ‘Kinase Glo’ plus Luminescent Kinase assay kit for the determination of IC₅₀ values. As for PknB, the reaction was carried out in a 96-well plate in 45 μl buffer (25 mM Tris-HCl pH = 7.4, 5 mM MgCl₂, 2 mM MnCl₂, 3 μM PknB) containing the compound. After incubation at 4 °C for 30 min, ATP was added to the reaction buffer at the final concentration of 100 μM. The assay was conducted at 37 °C for 3 hours. The intensity of luminescence signal was determined by Multilabel Plate Reader (PE Envision) with addition of 50 μl Kinase Glo reagent. The IC₅₀ values were calculated using the GraphPad Prism5 software.

PK activity was measured by using Kinase-Glo Plus Luminescent Kinase Assay kit (Promega Corporation, Madison, WI, USA). Purified WT or mutant PKM2 (50–100 nM) was added in 100 μl assay buffer containing 50 mM Tris pH 7.5, 100 mM KCl, 10 mM MgCl₂, 200 μM PEP, 200 μM ADP, and 3% DMSO. After a 15-min incubation, Kinase-Glo Plus reagent was added, according to the manufacturer’s instructions. In some cases, 0–40 μM FBP and 0–150 nM scFv 5 were added.

The enzymatic reaction catalyzed by Arc1, Bac1 and its mutants was assayed at 70°C by monitoring the increase in the amount of the product ATP using the Kinase-Glo Plus Luminescent Kinase Assay kit (Promega). The assay solution consisted of 50 mM HEPES (pH 8.0), 25 mM KCl, 10 mM (NH₄)₂SO₄, 2.0 mM Mg(CH₃COO)₂, 1.0 mM dithiothreitol, 1.0 mM ADP and 2.5 mM GTP. One enzyme unit equaled 1 μmol ATP formed per min. The Michaelis constant values (K_ms) for the substrate ADP and the catalytic rate constant (k_cat) were calculated based on the steady-state kinetic data with an assay solution of 50 mM HEPES (pH 8.0), 25 mM KCl, 10 mM (NH₄)₂SO₄, 2.0 mM Mg(CH₃COO)₂, 1.0 mM dithiothreitol, 2.5 mM GTP, with ADP at concentrations between 50 and 1000 μM. The kinetic parameters were calculated by nonlinear least-square fitting of the steady-state velocity data to the Michaelis-Menten equation using the Enzyme Kinetics module of SigmaPlot Ver. 13 (Systat Software).