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Assay Method Information

Assay Name:  Active p38alpha cascade assay
Description:  Inhibition of active p38a MAP kinase by inhibitors was determinedusing a p38a cascade activity assay. A 30-lL reaction mixture wasprepared containing 50 mM Hepes, pH 7.5, 1 mM EGTA, 10 mMMgCl2, 0.01% (w/v) Brij 35, 200 lM DTT, 3 lM Na3VO4, 1 mM ATP,100 nM unactivated MK2 and 60 pM active p38a. p38a kinase waspreincubated at room temperature with inhibitors for 30 min, followedby additions of ATP and the protein substrate MK2 (unactivated)to initiate the reaction. The reaction was run for 15 min atroom temperature. Ten microliters of 50 lM MK2-specific Omniapeptide substrate (Invitrogen, Carlsbad, CA, USA; Ser / Thr Peptide 3,cat no. KCZ1003) was then immediately introduced to the reactionmixture (final concentration = 10 lM) to determine the p38-activatedMK2 kinase activity. The Omnia peptide phosphorylation catalyzedby p38a-activated MK2 was followed by the increase influorescence at 485 nm (kex = 360 nm) over a 10-min period on aMolecule Device Fluorescence Plate Reader. Under such
Affinity data for this assay
 

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Last update November 1, 2007
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