Kinase assay

The kinase activity of wild‐type Src and Src mutants was determined by a luminescent ADP detection assay (ADP‐Glo™ Kinase Assay, Promega) using polyE₄Y and ATP as substrates. The phosphorylation reactions were performed in 50 μL volumes at 25°C in the protein kinase assay buffer: 50 mM N‐(2‐hydroxyethyl)piperazine‐N′‐3‐propanesulfonic acid (pH 8.0) containing 5% glycerol, 0.005% Triton X‐100, and 0.05% 2‐mercaptoethanol. The standard assay used 12 mM MgCl₂, 0.2 mM ATP, and 1 mg/mL polyE₄Y. After a 30 min incubation, 5 μL of the kinase reaction mixture was mixed with 5 μL ADP‐Glo™ reagent and incubated for 40 min to stop the kinase reaction and deplete the unconsumed ATP. Kinase detection reagent (10 μL) was added and the mixture was incubated for another 30 min to convert produced ADP to ATP and introduce luciferase and luciferin to detect converted ATP. Then the luminescence produced was measured by a plate‐reading luminometer. Assays were performed in duplicate, and each assay was repeated three times with reproducible results.