| Assay Method Information | |
| | EGFR kinase Enzymatic Activity Test 1 |
| Description: | To assess the effect of compounds on the activity of wild-type and mutant EGFR kinases, kinase activity was tested by measuring ATP consumption in an enzymatic reaction using an ADP-Glo (Promega Corporation) kit. Samples to be tested were dissolved in DMSO and diluted in a gradient. In a microplate, an EGFR kinase, a reaction buffer (containing Tris-HCl with a pH of 7.5, MgCl2, DTT and BSA), a kinase substrate Poly(Glu4, Tyr1) and a sample (a total volume of 20 μL per well) were added per well, while a blank control (no enzyme and sample) and a negative control (no sample) were set up; incubated for 15 minutes at 23° C.; added with 5 μL of ATP, and reacted at 23° C. for 60 minutes; added with ADP-Glo Reagent, and continuously reacted for 40 minutes at room temperature to inactivate the redundant ATP; then, added with a Kinase Detection Reagent, and reacted at room temperature for 30 minutes, then the chemiluminescence intensity L of each well was measured. The inhibition rate of the compound was calculated according to the value of the chemiluminescence intensity L, and the inhibition rate=[1-(Lsample−Lblank)/(Lnegative−Lblank)]×100%. IC50 values were calculated according to the above calculation using 4Parameter Logistic Model in XLfit software. |
| Affinity data for this assay | |
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