Assay Protocol ³³PanQinase™
The Assay
The ³³PanQinase™ assay is used at our German facility for compound screening on protein kinases.
Procedure Step-by-Step
In a ScintiPlate microtiter plate:
- Transfer 20 μl buffer
- Add 5 μl 10% DMSO (with or without test compound)
- Add 10 μl substrate (in 50 mM HEPES pH 7.5)
- Add 10 μl recombinant protein kinase
- Add 5 μl ATP (in H2O)
- Mix on shaker
- Incubate for 60 min at 30 °C
- Stop the reaction with 50 μl 2% H3PO4
- Mix on shaker
- Wash 3 times with 200 μl 0.9 % NaCl
- Count dry plate with a scintillation counter
The reaction takes place in ScintiPlate-96 (Cat. # 6005349, PerkinElmer), which are coated with a scintillant. Proteins and peptides bind to the surface of the plates, which allows us to use natural substrates in our assays. No artificial modification of the substrates is necessary.
Final Assay Concentrations
| 70 mM | HEPES-NaOH, pH 7.5 |
| 3 mM | MgCl2 |
| 3 mM | MnCl2 |
| 3 μM | Na-orthovanadate |
| 1.2 mM | DTT |
| ATP | variable, at apparent ATP-Km |
| [³³P-γ]-ATP | approx. 7-8 x 105 counts per minute |
| 1% (v/v) | DMSO (with or without compound) |
| Variable | Substrate |
| Variable | Kinase |
Variations
In addition, all PKC assays (except the PKC-mu and the PKC-nu assay) contain 1 mM CaCl2, 4 mM EDTA, 5 μg/ml Phosphatidylserine, and 1 μg/ml 1,2-Dioleylglycerol.
CAMK1D, CAMK2A, CAMK2B, CAMK2D, CAMK4, CAMKK1, CAMKK2, DAPK2, EEF2K, MYLK, MYLK2, and MYLK3 assays additionally contain 1 μg/ml Calmodulin and 0.5 mM CaCl2.
The PRKG1 and PRKG2 assays additionally contain 1 μM cGMP.
The DNA-PK assay additionally contains 2.5 μg/ml DNA.