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Assay Method Information |
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| | Kinase Assay |
| Description: | FlashPlates from Perkin Elmer (Boston, Mass., USA) with a 50 μl reaction volume are used. The reaction cocktail was pipetted in 4 steps in the following order: 15 μl of ATP solution (in H2O), 20 μl of assay buffer (see below), 5 μl of test sample in 10% DMSO, 10 μl of enzyme/substrate mixture (in H2O). The assay for all enzymes contained 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 μM Na-orthovanadate, 1.2 mM DTT, 50 μg/ml PEG20000, 1 μM [γ-33P]-ATP (approx. 8×10^5 cpm per well), protein kinase (variable amounts; see Table 1), and substrate (variable amounts). Certain assays also contained 1 mM CaCl2, 4 mM EDTA, 5 μg/ml Phosphatidylserine and 1 μg/ml 1.2-Dioleyl-glycerol. The MYLK2, CAMK1D, CAMK2A, CAMK2B, CAMK2D, CAMK4, CAMKK2, DAPK2 and EEF2K assays additionally contained 1 μg/ml Calmodulin and 0.5 mM CaCl2. The PRKG1 and PRKG2 assays additionally contained 1 μM cGMP. Recombinant Protein Kinases: All protein kinases were expressed in Sf9 insect cells or in E. coli as recombinant GST-fusion proteins or His-tagged proteins. All kinases were produced from human cDNAs, except JAK2, for which the mouse cDNA was used. Kinases were purified by affinity chromatography using either GSH-agarose (Sigma) or Ni-NTH-agarose (Qiagen). The purity of the protein kinases was examined by SDS-PAGE/coomassie staining. The identity of the protein kinases was checked by mass spectroscopy. Assays were made under license from Chemicon International Inc. for JAK2. The reaction cocktails were incubated at 30° C. for 80 minutes. The reaction was stopped with 50 μl of 2% (v/v) H3PO4, plates were aspirated and washed two times with 200 μl 0.9% (w/v) NaCl. All assays were performed with a BeckmanCoulter Biomek 2000/SL robotic system. Incorporation of 33Pi (counting of "cpm") was determined with a microplate scintillation counter (Microbeta, Wallac). |
| Affinity data for this assay | |
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