| Assay Method Information | |
| | Radiometric Assay |
| Description: | ALKl (Invitrogen, Carlsbad, CA, USA) kinase activity was measured at ProQinase GmbH ( Freiburg. Germany) in a radiometric assay using gamma-33P-ATP and casein (Sigma, St. Louis, MO, USA) as substrate in 96-weli PerkinElmer FlashPlates (Boston, MA, USA). The compounds were tested at 10 concentrations in the range of 1 x 10"4 M to 3 x 10"9 M in a total volume of 50 ul with a finalDMSO concentration of 1 % each. The assay components were mixed in the order: 20 ul assay buffer (70 mM HEPES-NaOH, pi I 7.5. 3 mM MgCl2, 3 mM MnCl2, 2uM sodium orthovanadate, 1.2 mM DTT); - 5ul gamma-33P-ATP (1.0ug/50ug in water, approx. 6 x10E+5 cpm per well); 5 ul test compound solution (in 10% DMSO);10 ul substrate (200 ug/ml, 1.0ug/50ml final concentration) / enzyme (4 ug/ml, 20 ng/50ul = 5.5 nM final concentration) solution (1 : 1 mixture). |
| Affinity data for this assay | |
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