Introduction
Kinases are involved in most cellular metabolic pathways.
They catalyze a phosphorylation reaction of their substrate in the presence of ATP.
Principle
The detection of the kinase activity can be performed by detection of the phosphorylation of the substrate, by quantification of residual ATP or by the quantification of generated ADP.
1. Detection of substrate phosphorylation: HTRF® KinEASE assay (CISBIO)
Two compatible fluorophores for TR-FRET (Time-Resolved Energy Transfer) assay, the donor europium cryptate and the acceptor XL665, are fused respectively to an antibody specific of the phosphorylation and to streptavidin which binds to the biotynylated substrate.
The TR-FRET signal between the fluorophores is proportional to the enzymatic activity.
2. Residual ATP determination: Kinase-GloTM assay (PROMEGA)
The enzymatic reaction is stopped by adding Luciferase and its substrate Luciferin. In the presence of Mg 2+ and ATP, Luciferase catalyzes the transformation of Luciferin into Oxyluciferin with emission of photon.
The reaction is quantified by measuring the luminescent signal which is proportional to the amount of ATP present in the reaction medium and thus inversely proportional to the enzymatic activity.
3. Determination of ADP produced by the reaction: ADP-GloTM assay (PROMEGA)
The enzymatic reaction is stopped by the addition of a reagent which removes the residual ATP.
A second reagent is then added to convert the ADP produced into ATP. Quantification of ATP performed using luciferin/luciferase reaction.
The luminescent signal measured is proportional to the amount of ADP produced by the enzymatic reaction.
4. Determination of ADP produced by the reaction: ADP-QuestTM assay (DISCOVERX)
The ADP formed is quantified by an enzymatic reaction involving pyruvate kinase and pyruvate oxidase.
ADP is converted into hydrogen peroxide, which is detected by a peroxidase in the presence of its substrate (Amplex Red), producing fluorescent Resorufin.
The fluorescence signal measured is proportional to the amount of ADP produced by the enzymatic reaction.
Protocol
ENZYMATIC REACTION | |||
|---|---|---|---|
| Format | 384 wells | ||
| Substrate + ATP +Kinase +/- inhibitor Reaction conditions to be defined | |||
| ENZYMATIC REVELATION | |||
| HTRF® KinEASE assay (CISBIO) | Kinase-Glo / ADP-Glo assays (PROMEGA) | ADP-Quest assay (DISCOVERX) | |
| Detection | Excitation wavelength : 337 nm Emission wavelength 1: 615 nm (donor fluorophore emission) Emission wavelength 2: 665 nm (acceptor fluorophore emission) | US Luminescence | Fluorescence measurement Excitation wavelength : 530 nm |
Notes :
- Allow 50 µL at 10 mM or 3 mg of sample to test 5 concentrations (3 points/concentration)
Inhibition of MSK-1 enzymatic activity
Determination of the kinetic parameters of a hiskinase
All enzymatic models can be adapted to your request.