Development of a time-resolved fluorescent assay for measuring tyrosine-phosphorylated proteins in cells |
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Authors: | Waddleton Deena Ramachandran Chidambaram Wang Qingping |
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Institution: | Department of Biochemistry and Molecular Biology, Merck Frosst Center for Therapeutic Research, P.O. Box 1005, Pointe-Claire Dorval, Que., Canada H9R 4P8. |
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Abstract: | We have developed a time-resolved fluorescent assay using Wallac's DELFIA system (DELFIA assay) to monitor changes in the phosphorylation level of insulin receptor from rat hepatoma (KRC-7) cells in response to ligand and the nonspecific, protein-tyrosine phosphatase inhibitor pervanadate. In this system, a biotinylated antiinsulin receptor antibody was used to capture the insulin receptor and an europium-labeled antiphosphotyrosine antibody was used to assess tyrosine phosphorylation. This assay provides a highly sensitive, nonradioactive readout of receptor phosphorylation. We have validated the DELFIA assay by directly comparing receptor phosphorylation using the well-established technique of immunoblotting. The utility of the DELFIA assay in measuring the phosphorylation status of other receptors has also been demonstrated using epidermal growth factor receptor from A431 cells. |
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Keywords: | Insulin receptor Tyrosine phosphorylation Protein-tyrosine phosphatases DELFIA assay Epidermal growth factor receptor KRC-7 cells A431 cells |
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