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| | Cell-Free Kinase Assays |
| Description: | The inhibitory effect of the compounds according to the invention was tested on various serine/threonine, tyrosine and lipid kinases in enzymatic assays. Recombinant human kinases such as, for example, Erk2, were used in this case, partly as full-length kinases, partly as shortened fragments, but at least consisting of the functional kinase domains. The commercial kinase proteins (Proqinase, Upstate) were used as recombinant fusion proteins with GST (glutathion-S-transferase) or His-Tag. Depending on the type of substrate, the various kinase reactions were quantified by means of suitable ALPHA beads (Perkin-Elmer). MAPK-ALPHAs (e.g. Erk2): the test substance, 0.625 ng Erk2 (#14-173, Upstate), 10 uM ATP and 15 nM biotinylated MBP (myelin basic protein) substrate were incubated on a 384-well Optiplate (Perkin-Elmer) in a volume of 15 ul for 1 h in 25 mM Tris, 10 mM MgCl2, 0.1% Tween-20, 100 uM NaVO4, 2 mM DTT at pH 7.5. |
| Affinity data for this assay | |
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