Enzychrom kinase assay kit

The kinase activity assay was performed using the EnzyChrom Kinase Assay Kit (BioAssay Systems, USA) to test the differences in protein kinase activity before and after CoCl₂ treatment in vitro. Detailed information about kinase activity assay is provided in the Supplementary Materials and Methods.

For kinase assay of the SnRK2.1, autophosphorylation kinetics of SnRK2.1 purified from soluble and insoluble fraction were measured by using the EnzyChrom™ Kinase Assay Kit (BioAssay Systems, Hayward, US), respectively. Kinase activity was measured by the quantification of ADP, which was enzymatically converted to ATP and pyruvate, using a fluorimetric (530 nm/590 nm) assay method. 0.5 μg purified SnRK2.1 was incubated in 32 μL reaction buffer provided by the kit with 150 μM ATP at 20°C for the indicated time in each kinase assay (40 μL reaction volume). As the blank control, the reaction buffer mixed with 150 μM ATP without enzyme was also incubated for the indicated time. After incubation with the working agent for 10 min, the fluorescence intensity was read at λexc = 530 nm and λem = 590 nm. The standard curve was performed as indicated by the kit. The kinase activity was calculated as the instruction of the EnzyChrom Kinase Assay Kit. Specifically, Kinase Activity = (ΔF_SAMPLE/(Slope · t)) × (40 μL/Vol (μL)) (U/L), where ΔF_SAMPLE = (fluorescence intensity of sample well − fluorescence of the blank well); slope is the slope of the ADP standard curve. t is the kinase reaction time (5 min was adopted here). Vol is the volume (μL) of kinase added to the 40 μL reaction.