| Assay Method Information | |
| | Biochemical Assay (1 uM) |
| Description: | A typical assay was conducted in 20 μL volume including 60 μM active p38α (Carna, cat #04-152), 10 μM ATP, 1 μM FITC-HSP27 peptide (Sangon, Cat #P22354), and 1 nM inactive MK2, or PRAK in 1× reaction buffer (20 mM HEPES, pH7.5, 10 mM MgCl2, 1 mM DTT, 0.0100 Triton X-100, 0.01% BSA). After 2 h incubation of the reaction mixture with various concentration of invention compound (200 nL), 60 μL 1×IMAP solution Mixture (Molecular Devices, Cat #R8127) was added to the reaction mixture and incubated for another half hour. The signal was then read by Synergy Neo2 Multi-Mode Microplate Reader with filter setting (Ex/Em=485 nm/FITC FP-P pol 528 nm & FITC FP-S pol 528 nm). The signal was then normalized to vehicle control and fitted in Xfit to generate IC50. The selectivity of MK2 over PRAK was calculated by the formular Selectivity=IC50 of PRAK/IC50 of MK2. |
| Affinity data for this assay | |
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