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Kinase glo luminescent kinase assay

Manufactured by Promega
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The Kinase-Glo® Luminescent Kinase Assay is a biochemical assay used to measure kinase activity. The assay quantifies the amount of ATP remaining in a kinase reaction, which is proportional to the kinase activity.

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12 protocols using kinase glo luminescent kinase assay

1

ROCK Kinase Inhibition Assay

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The ROCK kinase assay is a luminescent kinase assay that measures IOP formed from a kinase reaction. ADP is converted into ATP and is converted into light by Kinase-Glo Luciferase. The Kinase-Glo Assay can be used to monitor the activity of virtually any ADP-generating enzyme. The relative light unit (RLU) without test compound was set as 100% (blank value), and that without enzyme and compound was set as 0% (normal value). The reaction rate (% of black) was then calculated from the RLU.
The following reagents were used in this assay: ROCK1 (750 ug/mL, Carna Biosciences, Cat.01-109); long S6 kinase substrate peptide (Millipore, Cat#12-420); magnesium chloride solution (Sigma, 60142-100 mL); EGTA solution (0.5 M pH 8.0, Bioword, Cat. 4052008-1); Trizma® hydrochloride solution (1 M, pH 7.5 Sigma, T2319-100 mL); bovine serum albumin solution (20 mg/mL in water, Sigma); Kinase-Glo luminescent kinase assay (Promega, RV6712); ripasudil (K115) hydrochloride dehydrate (Selleckchem, S7995-5 mg); ATP solution (100 mM, GE Healthcare, Cat.27-2056-010). A ROCK kinase inhibitor k-115 was used as the positive control.
Inhibition (%)=100 {(activity of Enzyme with Test Cmpd & SbustrateMin)}×100MaxMin
Max = observed enzyme activity measured in the presence of enzyme, substrate(s), and cofactors utilized in the method.
Min = Normal value in the method
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2

Kinase Activity Assay for miRNA Effects

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A total of 3000 exponential-phase cells were plated into each well of a 96-well plate (100 μL medium/well) and cultured overnight. Then, the cells were transfected with miR-338 mimics or control mimics, inhibitor RNA or control RNA for another 48 h. The assays were conducted with a Kinase-Glo® Luminescent Kinase Assay (Promega) according to the manufacturer's protocol. Data were expressed as fold-changes.
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3

Kinase Enzyme Kinetics Assay Protocol

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Enzyme kinetics was measured by Kinase-Glo Luminescent Kinase Assay and ADP-Glo Kinase Assay (Promega, WI, United States). The 5 μl of different concentrations of LM49 solution, 2.5 μl different concentrations of ATP solution, 2.5 μl of GSK3β solution, and 10 μl Kinase-Glo Reagent were mixed and incubated in a well of a solid white 96-well plate for 30 min at 30°C. Then, 20 μl of ADP-Glo Reagent was added to the well and incubated at room temperature for 40 min. Finally, 40 μl of Kinase Detection Reagent was added to the well and incubated at room temperature for 30 min. Luminescence was measured using a SpectraMax i3X multifunctional microplate reader (Molecular Devices, CA, United States). Lineweaver-Burker plot was generated by 1/V and 1/[ATP].
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4

ATP Depletion Kinase Activity Assay

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GST-DYRK1 fusion proteins were aliquoted at amounts that resulted in the depletion of approximately 90% of the ATP in the Kinase-GLO Luminescent Kinase Assay from Promega (linear range of the kinase titration curve). These aliquots were incubated for 3.5 min at temperatures between 39 °C and 47 °C as indicated in Figs. 2C and S2. Immediately thereafter, assays were run in a total volume of 15 μL with kinase-buffer (25 mM Hepes pH 7.4, 0.5 mM DTT, 5 mM MgCl2) in the presence of 5 µM ATP and 20 μM DYRKtide as a substrate peptide. Reactions proceeded at room temperature for 30 min before the luciferase reaction was run to determine the ATP depletion by the kinase reaction. The decrease in ATP levels after the reaction was used as a measure of catalytic kinase activity.
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5

Kinase Activity Assay using Luminescence

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Kinase activity in the presence of Mef or IKK‐16 was measured using a Kinase‐Glo Luminescent Kinase Assay (Promega) as described previously.20
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6

Measuring cGAS cGAMP Processing Activity

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cGAMP processing ability of cGAS in vitro was measured with Kinase-Glo® Luminescent Kinase Assay (Promega). Detailed product catalog is listed in Supplementary Table 1.
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7

Screening Compounds for ALK2 Inhibition

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The initial screening to
identify candidate compounds was performed by the Kinase-Glo Luminescent
Kinase Assay (Promega, Madison, WI). The reaction buffer contained
10 mM HEPES (pH 7.4), 150 mM NaCl, 20 mM MgCl2, and 0.5%
dimethyl sulfoxide (DMSO). The assay mixture contained 1 μM
purified ALK2 (R206H) kinase domain (aa. 201-499), 10 μM substrate
peptide, 5 μM ATP, and 4 μM each compound. The substrate
peptide used was NPISSVS, designed and synthesized to be homologous
to the phosphorylated Smad sequence. First, the protein was incubated
with each compound for 30 min, and then the kinase reaction was started
and continued for 30 min. The efficacies of the compounds were determined
by the remaining amounts of ATP, quantified by the luminescent signal
after the reaction.
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8

Inhibitory Effects of Chinese Medicine Monomers on VicK Protein ATPase

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The inhibitory activities of the Chinese medicine monomers for the ATPase activity of the full-length VicK protein were evaluated using the Kinase-Glo™Luminescent Kinase Assay (Promega, Madison, USA). Briefly, 6 μg VicK protein was pre-incubated with a series of dilution of compounds in the reaction buffer at 37°C for 30 min. 5 μM ATP was added and incubated for another 30 min. Kinase-Glo™Reagent was also added to detect the remaining ATP recorded from luminescence measurements (RLU). In parallel, the VicK protein with no addition of compounds was used as a control, and the mixture with compounds excluding the VicK protein served as another control. The rate of protein phosphorylation (Rp) inhibition by the compounds was calculated from Equation (1).
The half maximal inhibitory concentration (IC50), which is the concentration of inhibition of 50% VicK protein autophosphorylation, was determined by GraphPad Prism 5 (Qin et al., 2006 (link)).
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9

Inhibition of YycG Histidine Kinase Activity

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In order to verity interaction of two thiazolidione derivatives (H2-60 and H2-81) with the HATPase_c domain of YycG, impact of these two derivatives on the ATPase activity of the YycG′ protein were measured using the Kinase-Glo™ Luminescent Kinase Assay (Promega, Madison, USA). Briefly, 3 μg purified YycG′ protein was pre-incubated with a series of dilutions of the compounds in reaction buffer [40 mM Tris (pH 7.5), 20 mM MgCl2 and 0.1 mg/ml BSA] at room temperature for 30 min, 4 μM ATP was added and incubated for 30 min at room temperature, then Kinase-Glo™ Reagent was added to detect the remaining amount of ATP, and the results were reflected by luminescence intensity value (RLU). Meanwhile, YycG′ protein with no addition of the derivatives was used as the control and ATP only was used as a blank. The rate of inhibition of kinase phosphorylation (Rp) by the derivatives was calculated by the following equation: Rp=RLUYycG'+derivatives+ATP-RLUYycG'+ATPRLUATPonly-RLUYycG'+ATP×100%
IC50 (the concentration resulting in 50% inhibition of YycG′ histidine kinase auto-phosphorylation) was obtained by using GraphPad v7.0 software (San Diego, CA, USA). BSA protein without phosphorylation activity were used as the negative control. All experiments were performed in triplicate.
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10

Measuring ATP Hydrolytic Activity

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ATP hydrolytic activity of proteins was measured using Kinase-Glo® Luminescent Kinase Assay (Promega, United States). A 50 µl reaction mixture (4 µg protein, 2 µM ATP, 40 mM Tris, pH 7.5, 20 mM MgCl2, 0.1 mg/ml BSA) with drugs (20 µg/ml 666-15 or 1.25 µg/ml PB alone or 1.25 µg/ml PB + 5, 10, 20 µg/ml 666-15) was incubated for 1 h at 37°C within a 96-well plate, and then mixed with 50 µl substrate reagent and incubated for 10 min at room temperature. The luminescence signal was recorded using an Infinite M200 PRO Multimode Microplate Reader (Tecan).
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