Assay Protocol PanQinase

Assay Protocol ³³PanQinase™

The Assay

The ³³PanQinase™ assay is used at our German facility for compound screening on protein kinases. 

Procudure Step-by-Step

In a FlashPlate 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 reaction with 50 μl 2% H3PO4
  • Mix on shaker 
  • Wash 3 times with 200 μl 0.9 % NaCl 
  • Count dry plate with scintillation counter

The reaction takes place in FlashPlates (Cat. # SMP200 or SMP400, 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.

The 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.