Development of a time-resolved fluorescent assay for measuring tyrosine-phosphorylated proteins in cells.
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.
Waddleton
Deena
D
Department of Biochemistry and Molecular Biology, Merck Frosst Center for Therapeutic Research, P.O. Box 1005, Pointe-Claire Dorval, Que., Canada H9R 4P8.
Ramachandran
Chidambaram
C
Wang
Qingping
Q
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D019921:Biotinylation; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D005063:Europium / Q000032:analysis / Q000378:metabolism; D015200:Fluoroimmunoassay / Q000379:methods*; D007328:Insulin / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis* / Q000378:metabolism; D011233:Precipitin Tests; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D051381:Rats; D011972:Receptor, Insulin / Q000032:analysis* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D007328:Insulin; D019000:Phosphotyrosine; D005063:Europium; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D017027:Protein Tyrosine Phosphatases