ELISA-Based Kinase Assay: Inhibition of erbB tyrosine kinase activity was assessed using an ELISA-based receptor tyrosine kinase assay. Kinase reactio...

ID: ALA3705968

Type: Binding

Description: ELISA-Based Kinase Assay: Inhibition of erbB tyrosine kinase activity was assessed using an ELISA-based receptor tyrosine kinase assay. Kinase reactions (50 mM HEPES, pH 7.4, 125 mM NaCl, 10 mM MgCl2, 100 μM sodium orthovanadate, 2 mM dithiothreitol, 20 uM ATP, test compound or vehicle control and 1-5 nM GST-erbB per 50 uL reaction) were run in 96-well plates coated with 0.25 mg/ml poly-Glu-Tyr (Sigma). The reactions were incubated for 6 minutes at room temperature while shaking. Kinase reactions were stopped by removal of reaction mixture, then wells were washed with wash buffer comprising 3% Bovine Serum Albumin and 0.1% Tween 20 in Phosphate Buffered Saline (PBS). Phosphorylated tyrosine residues were detected by adding 0.2 μg/ml anti-phosphotyrosine antibody (Oncogene Ab-4; 50 μL/well) coupled to Horse Radish Peroxidase (HRP) for 25 minutes while shaking at room temperature.

Format: BAO_0000357

Organism: Homo sapiens

Target:  Epidermal growth factor receptor erbB1(ALA203)

Document:  ALA3639119

BindingDB Source:  6153