| Assay Method Information | |
| | PKC-Theta and PKC-Delta Inhibition Assay |
| Description: | PKC-theta and PKC-delta biochemical activities were measured using the PKC-theta HTRF KinEASEkit kit, according to manufacturer's instructions (Cisbio, catalogue number 61ST1PEJ). Briefly, the kinase buffer component of the kit was supplemented with 10 mM MgCl2, 1 mM DTT and 0.1% Tween 20. For the PKC-theta assay, STK substrate and ATP were added to provide a final assay concentration of 525 nM and 6.5 μM, respectively. For the PKC-delta assay, STK substrate and ATP were added to provide a final assay concentration of 243 nM and 5.7 μM, respectively. The streptavidin_XL665 and STK antibody-cryptate detection reagents were mixed according to the manufacturer's instructions. Test compounds were diluted in DMSO in a series of 10 semi-log step doses; 10 nL of each compound dose were dispensed in 384 well plates. Recombinant human PKC-theta (His-tagged 362-706) or PKC-delta (His-tagged 345-676) was diluted into kinase buffer to provide a final assay concentration of 10 ng/ml and added to the test compound for 30 minutes on ice. The reaction was started by addition of the substrate and ATP and incubated at 25° C. for 30 minutes or 20 minutes for the PKC-theta and PKC-delta assays, respectively. The detection reagents were added, and the plate was incubated in the dark for 2 hours. Fluorescence was measured on an Envision 2103 plate reader with optical setup for excitation at 665 nM and emission at 620 nM in the HTRF mode. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |