Hydroxylapatite “batch” assay for estrogen receptors: Increased sensitivity over present receptor assays

• Published in: Journal of steroid biochemistry Vol. 7; no. 5; pp. 357 - 368
• Main Authors: Pavlik, E.J., Coulson, P.B.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 01.05.1976
• Subjects: Animals; Binding, Competitive; Estradiol - metabolism; Female; Hydroxyapatites; Kinetics; Methods; Muscle Proteins - isolation & purification; Muscle Proteins - metabolism; Rats; Receptors, Cell Surface; Uterus - metabolism
• ISSN: 0022-4731
• DOI: 10.1016/0022-4731(76)90095-9

A “batch” hydroxylapatite procedure for the adsorption of the uterine estradiol 17β-receptor complex is described. Characterization with respect to washing efficiency, binding specificity, competition, adsorption time, sensitivity and stability against increasing KC1 ionic strength were included. Equilibrium parameters obtained by Scatchard analysis were compared to the range of values found in the literature. K ta and receptor site concentration per uterus obtained by this “batch” technique were found to be well within the range described by these reported values. This technique is particularly advantageous due to its wide range of operational sensitivity (capable of detecting specific estradiol-17β binding to a cytosol fraction containing from 5 to 500 μg protein per 225 μl). The assay is run entirely at low temperature (0–2°C). In addition this technique depends on a homogeneous insoluble chemical, hydroxylapatite, which can be obtained in analytical grade quantities of uniform particle size, shows little affinity for free steroid, can be readily packed or resuspended, and appears independent of changes in concentrations of KC1 up to 2500 mM. Additional considerations include the effect of temperature during assay, the importance of empirical correction for non-specific binding, the contributions of binding information on the calculation of equilibrium parameters and statistical evaluation of random error and assay repeatability.