| Assay Method Information | |
| | Flashplate Assay |
| Description: | In vitro enzyme inhibition using scintillation of incorporated radio label (flashplate assay). Test compounds diluted in DMSO are mixed with a suitable substrate / co-substrate (here: bio- tinylated a-casein and 3P-ATP) in a corresponding assay buffer. Addition of the enzyme of interest (here: ALKl kinase) starts the enzyme reaction. The enzyme-catalyzed incorporation of radio label into the substrate is measured via scintillation. Incorporated radio label is separated from free radio label via specific binding of the biotinylated substrate to strepavidin-coated microliter plates (flashplates) and concomitant washing steps. The scintillation signal intensity (counts per minutes, cpm) is proportional to the enzyme activity. Enzyme inhibition results in a decreased signal intensity. IC50 values of the test compounds are determined by cpm-versus-[I] plots. Reaction buffer: Reaction buffer contains 50 mM Tris pi I 8.0 (Sigma), 1 mM MnCi2 (Sigma), 0.01% Nonidet P40 (Fluka). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |