New, simple insulin-receptor assay with universal application to solubilized insulin receptors and receptors in broken and intact cells.
A new, simple insulin-receptor-binding assay has been devised. The assay is based on the separation of free and receptor-bound 125I-labeled insulin in 80% ethanol. It was found that the insulin-receptor complex was fully stable at this ethanol concentration, regardless of the source of the receptor employed. The assay has been evaluated with solubilized insulin receptors and membrane-bound receptors from human placenta and porcine liver as well as intact cells with the IM-9 cell line. The assay is simple, rapid, and has large capacity. Comparisons of the ethanol-based assay to the conventionally employed assays with polyethylene glycol or microfuge centrifugation for the separation of free and bound 125I-insulin revealed large discrepancies between the assays. The ethanol-based assay always appeared to provide a better separation. Microfuge centrifugation of placental membranes precipitated approximately 3% of the ethanol-precipitable insulin-receptor complex, while polyethylene glycol precipitation of solubilized insulin receptors varied between 40 and 80% of the ethanol precipitability, depending on the receptor concentration employed.
D002462:Cell Membrane / Q000378:metabolism; D002498:Centrifugation; D011232:Chemical Precipitation; D000431:Ethanol; D006801:Humans; D007328:Insulin / Q000378:metabolism; D008099:Liver / Q000032:analysis; D008214:Lymphocytes / Q000032:analysis; D010920:Placenta / Q000032:analysis; D011092:Polyethylene Glycols; D011972:Receptor, Insulin / Q000032:analysis*
D007328:Insulin; D000431:Ethanol; D011092:Polyethylene Glycols; D011972:Receptor, Insulin