IGF1-R Cellular Phosphorylation Assay (intracellular kinase activity assay) for compound screening and profiling in intact cells
The mitogenic signaling in mammalian cells is carried out mainly by growth factors that interact with receptors localized at the plasma membrane. Most of these receptors have a tyrosine kinase activity domain that is localized at the cytoplasmic region of the molecule. The interaction of the growth factors with the receptors, besides inducing the kinase activity of the receptor, activates signaling pathways that alter gene expression patterns and induce mitogenesis, or if deregulated are related to cancer. Among these receptors IGF1-R has been characterized as target for directed therapy.
IGF1R
JTK13
MEF
Transfected
In the cellular IGF1-R phosphorylation assay the murine embryonal fibroblast cell line (MEF) is used, which expresses a high level of exogenously introduced full-length human IGF1-R. Stimulation of these cells with the physiological ligand human Insulin like growth factor 1 (IGF1), results in a robust IGF1-R autophosphorylation. Compounds are preincubated before cell stimulation to allow thorough target binding. Stimulation conditions are optimized to determine dose-related inhibition of the phospho-IGF1-R signal, which is subsequently quantified by Sandwich-ELISA technique. The assay is validated based on known inhibitors of IGF1-R kinase activity (see. Fig.1).
Substrate phosphorylation as a readout of intracellular kinase activity via ELISA
Freiburg, Germany
More information can be found on our website Cellular Phosphorylation Assay Services.
Reference compound IC50 for IGF1-R
Staurosporine blocks IGF1-R as a broad kinase inhibitor and inhibits the cellular phospho-IGF1-R response with highly reproducible IC50 values. The graph shows a representative result.