| Assay Method Information | |
| | Kinase Assay |
| Description: | 1. Experiment PurposeTo determine the inhibitory activity of compounds against kinases by the kinase assay kit ADP-GLO.2. Experiment PrincipleDRAK2 is a serine/threonine protein kinase. ADP Glo& #8482; Kinase Assay (ADP Glo& #8482; Kinase Assay Kit) is a luminescent kinase assay kit that can detect ADP formed during kinase reactions; this kit can firstly convert ADP into ATP, which is then captured by Ultra Glo& #8482, luciferase and ultimately converts into light. The emitted light signal is positively correlated with the corresponding ATP, thus through this method, the inhibitory activity of small molecule compounds on DRAK2 can be measured.3. Experimental SampleThe compound was dissolved in DMSO before experiment to prepare stock solution.When using, the stock solution was diluted with culture medium to a desired concentration.4. Experimental MethodsI. Kinase Assay1. 1 μl of compound was added to the well for each assay2. 2 μl of kinase solution was added to each well of the assay plate, except for the control well that does not contain enzymes (changed to add 2 μL of 1× kinase buffer).3. 2 μl of ATP was added to each well of the assay plate4. the plate was shook and centrifuged.II. Stop Measurement2.5 μl ADP-Glo reagent was add to terminate kinase reaction and consume unconsumed ATP, leaving only ADP and very low ATP background to incubate at room temperature for 60 minutes.III. Assay Analysis5 μl of kinase assay reagent was add to convert ADP to ATP, and luciferase and fluorescein were introduced to detect ATP.IV. Data ProcessingEnvision was used to measure luminescenceV. Curve Fitting1. Values were copied from Envision program2.Inhibitionpercentage= (maximumsamplingrate)/(maximum-minimum)X100.The minimum refers to the proportion of without enzyme control, while maximum refers to the proportion of DMSO control.3. GraphPad Prism 5.0 was used to process data |
| Affinity data for this assay | |
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