| Assay Method Information | |
| | PIM2 Activity Assay |
| Description: | Kinase Buffer (KB) The buffer for PIM2 assay was composed of HEPES 50 mM, at pH 7.5, with 1 mM MgCl2, 1 mM DTT, 3 uM Na3VO4, and 0.2 mg/mL BSA.Full-length human PIM2 was expressed and purified as described in Fedorov O, et al., PNAS 2007 104, 51, 20523-28.ii. Assay Conditions (Final Concentrations)Enzyme concentration=1.5 nMAktide substrate (Chemical Abstract Service Registry Number 324029-01-8)=5 uMATP=4 uM 33P-gamma-ATP=1 nMiii. Robotized Dowex AssayThe test mix consisted of: 1) 3x Enzyme mix (done in Kinase Buffer 3x), 5 uL/well 2) 3x substrate and ATP mix (done in ddH2O), together with 33P-gamma-ATP, 5 uL/well 3) 3x test compounds (diluted into ddH2O-3% DMSO)-5 uL/well. |
| Affinity data for this assay | |
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