Epidermal growth factor (EGF) binding to membranes immobilized in microtiter wells and estimation of EGF-related transforming growth factor activity.
The binding of radiolabeled epidermal growth factor (EGF) to immobilized A-431 target cell membranes coupled to polyvinyl chloride microtiter wells is described. Saturation curves and Scatchard analysis of the data indicate that the observed binding parameters are consistent with those previously reported. Binding capacity of the membranes are approx. 6.6 pmol EGF per mg membrane protein. Kinetics of 125I-EGF binding were slower, however, than reported for binding to membranes in suspension, although binding constants were not greatly different. The high- and low-affinity binding constants for 125I-EGF were calculated to be approximately 1 X 10(12) M-1 and 2.5 X 10(9) M-1, respectively. Application of this technique in a competitive binding assay requires no more than 2.5 micrograms of membrane protein per assay, is essentially complete after 60 min, and facilitates screening of a large number of samples in a short time. Therefore, this will assist in the evaluation and quantitation of EGF and EGF-related transforming growth factor activity in physiological fluids. This technique may also be applied to analyses of other hormone-receptor systems.
D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors; D006801:Humans; D007700:Kinetics; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D015291:Transforming Growth Factors
D010455:Peptides; D011956:Receptors, Cell Surface; D004815:Epidermal Growth Factor; D015291:Transforming Growth Factors; D066246:ErbB Receptors