| Assay Method Information | |
| | HCK Kinase Assay |
| Description: | HCK kinase reaction (10 μL) containing 4 nM N-terminally GST-tagged HCK (75-526), purified from insect expression system, 5 μM Control AMC Substrate, 2 μM Src-Family Kinase R110 Substrate, and 50 μM ATP in kinase reaction buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA, 1 mM MnCl2, 0.1 mM Sodium Vanadate), and test compound 1:3 serially-diluted starting at 1 μM were incubated at room temperature (22-25° C.) for 60 minutes in 384 well plate (Corning, Cat. No. 4514). The procedure of ProFluor Src-Family Kinase Assay (Promega, Cat. No. V1271) was then followed. To the reaction was added 5 μL Protease solution and the mixture was incubated for 60 minutes at room temperature (22-25° C.), followed by 5 μL Stabilizer solution. The fluorescence signal was read on an Envision multilabel plate reader (Perkin Elmer). The R110 was then read at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The AMC signal was read at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. |
| Affinity data for this assay | |
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