| Assay Method Information | |
| | Unactivated p38alpha cascade assay |
| Description: | Binding of inhibitors to unactivated p38a that leads to decreasedphosphorylation of p38alpha by MKK6 was determined by measuringthe activated p38alpha activity in the assay mixture with andwithout inhibitors. In a 20-lL reaction mixture containing 50 mMHepes, pH 7.5, 1 mM EGTA, 10 mM MgCl2, 0.01% (w / v) Brij35 and 200 lM DTT, unactivated p38a (2 nM) was preincubatedwith inhibitors at various concentrations for 30 min at roomtemperature. Ten microliters of 1 mM ATP and 1 nM active MKK6were then introduced to initiate p38a activation. The reaction wasrun at room temperature for 15 min. The mixture was thenimmediately diluted 100-fold so that the inhibitor concentrationwas reduced by 2 orders of magnitude and residual inhibitoryeffect was minimized. The reaction product, active p38alpha, wasthen calculated based on its catalytic activity to activate MK2 asdescribed in Active p38a cascade assay section. |
| Affinity data for this assay | |
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