| Assay Method Information | |
| | LANCE® Eu Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Kinase Assay |
| Description: | LANCE® Eu time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assays (PerkinElmer) were performed in 384-well OptiPlates (Corning) using recombinant AMPK(α1) and AMPK(α2) kinase subunits (Carna), ULight™-CREBtide substrate (PerkinElmer) and ATP (Sigma) according to supplier protocols. All reagents were prepared in kinase buffer (2 mM DTT, 50 mM HEPES, 1 mM EGTA, 10 mM MgCl2, 0.01% Tween20, pH 7.5) and inhibitor solutions were prepared such that the final DMSO concentration did not exceed 0.5%, which was shown to have no effect on kinase activity. AMPK(α1) and AMPK(α2) were used at a final concentration of 4 nM, ULight™-CREBtide substrate was used at a final concentration of 50 nM and ATP was used at final concentration of 8 μM and 20 μM for AMPK(α1) and AMPK(α2), respectively. Assays were performed at 25° C. in a reaction mixture consisting of 2.5 μL serially diluted inhibitor solution, 2.5 μL kinase, 2.5 μL ATP and 2.5 μL substrate. Reagents were incubated for 1 hr before the reaction was halted through the addition of EDTA (10 mM) after which Eu anti-phospho-CREB (Ser133) antibody (PerkinElmer) was added at a final concentration of 2 nM for 1 hr. The plate was read using a BioTek Synergy H1 Hybrid plate reader enabled for TR-FRET (Excitation=340 nm; Substrate emission=665 nm; Antibody emission=615 nm; Delay=100 μs; Integration=200 μs). Emission ratios (665 nm/615 nm) were calculated for each well and half-maximal inhibitory concentration (IC50) values were determined for each inhibitor through non-linear regression analysis of the log dose-response curve. LanthaScreen™ Eu TR-FRET assay (Invitrogen) was performed in 384-well low volume plates (Corning) using recombinant KDR kinase (Carna), Kinase Tracer 236 (Invitrogen) and LanthaScreen™ Eu-anti-GST antibody (Invitrogen). KDR was used at a final concentration of 5 nM, Kinase Tracer 236 was used at a final concentration of 150 nM and LanthaScreen™ Eu-anti-GST antibody was used at a final concentration of 2 nM. All reagents were diluted in 1× kinase buffer A (Invitrogen) and assays were performed at 25° C. in a reaction mixture consisting of 5 μL serially diluted inhibitor solution, 5 μL Kinase Tracer 236 solution, and 5 μL kinase/antibody solution. |
| Affinity data for this assay | |
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