| Assay Method Information | |
| | Biochemical Assay |
| Description: | This study evaluates the inhibitory potency of invention compound on p38/MK2 pathway vs p38/PRAK pathway. More specially the compound concentration (IC50) was determined that inhibited half of the maximal activation of MK2 or PRAK by p38. MK2 activation study was set up without or with a serial of 10-point 1:3 dilution of invention compound at top dose of 1 or 1 μM, and PRAK activation study was set up without or with a serial of 10-point 1:3 dilution of invention compound at top dose of 300 μM. The MK2 and PRAK activity were determined by the phosphorylation level of HSP27 peptide conjugated with FITC. A typical assay was conducted in 20 μL volume including 60 pM active p38a (Carma, 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 | |
|---|---|
| If you find an error in this entry please send us an E-mail | |