Methodology:
All ATP producing and ATP degrading enzymatic reactions can be continuously monitored by measuring the light from firefly luciferase/luciferin being present in the reaction mixture. Examples include protein kinases67, adenylate kinase14, pyruvate kinase6, pyruvate phosphate dikinase, glycerol kinase43, 44, creatine kinase isoenzymes8, 10, 18, 22, ATPases14, ATP synthetases49, aminoacyl tRNA synthetase. Substrates to such enzymes can be measured kinetically or with endpoint assays. Examples include ADP, AMP, pyrophosphate. When continuously monitoring ATP the luciferase ATP depletion should be negligible compared to the enzymatic reactions being measured.  ATP concentrations from 1 to 1,000,000 pmol/L can be monitored. ATP depletion assays are best set up as 1st order reactions plotting the natural logarithm of the light versus time. The slope in such plots is the rate constant of the reaction. ATP forming reactions can be calibrated by measuring the light before and after adding a known amount of ATP standard at the end of each test.

Intended use:

    • Monitoring of ATP forming and ATP degrading enzymatic reactions.
    • Assays of enzymes and metabolites that can be coupled to ATP formation or degradation.
    • Monitoring of cell lysis (ATP or ATP producing enzymes)
    • Monitoring of oxidative phosphorylation
    • Monitoring of photophosphorylation

Suitable products:
ATP Kit SL, Kinase RR Kit, ATP Standards

Recommended paper:
Saint-Léger, A, Ribas de Pouplana, L (2017) A new set of assays for the discovery of aminoacyl-tRNA synthetase inhibitors. 113, 34-45

Lundin, A. Jäderlund, B. and Lövgren, T. (1982) Optimized bioluminescence assay of creatine kinase and creatine kinase B-subunit activity. Clin. Chem. 28, 609-614.

Lundin A. (2014) Optimization of the firefly luciferase reaction for analytical purposes. Adv. Biochem. Engin./Biotechnol. 2014; 145:31-62

 

Lundin, A. (2000) Use of Firefly Luciferase in ATP-Related Assays of Biomass, Enzymes, and Metabolites. Methods in Enzymology, 305, 346-370.

Please choose the kit best describing your assay.

111-021

Kinase RR Kit

2596kr

Identification of protein kinase inhibitor

Kinase RR Kit or Kinase Reaction rate Kit is developed for the easy and reliable identification of protein kinase inhibitor candidates from large compund libraries. The Kinase RR Kit uses direct bioluminescent measurement of ATP degradation rate to distingusih hits from non-hits. In contrast to other bioluminescent ATP based assays of kinase there is no interference from inhibitors acting on luciferase. Furthermore the standard curve is linerar rather than sigmodial. the Kinase RR Kit is flexible and can easily be adapted to various HTS strategies. Z’ values up to 0.96 have been reported. The assay does not require radioactive materials, antibodies or any form of conjugates.

144-041

ATP Kit SL

3207kr

For assays of both enzymes and metabolites

ATP Kit SL is intended for assays of enzymes and metabolites by monitoring the formation or degradation of ATP (adenosine triphosphate) over the range of 10 –12 to 10 –6 mol/L.