Hydrophobic interaction chromatography (HIC) for the separation of protein-bound and free steroids. Application to binding protein and receptor assays

Détails

ID Serval
serval:BIB_E7C386F76ABC
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
Hydrophobic interaction chromatography (HIC) for the separation of protein-bound and free steroids. Application to binding protein and receptor assays
Périodique
Journal of Steroid Biochemistry
Auteur(s)
Steimer  T., Theintz  G. E., Sizonenko  P. C., Herrmann  W. L.
ISSN
0960-0760
0022-4731 (Print)
Statut éditorial
Publié
Date de publication
12/1985
Volume
23
Numéro
6A
Pages
955-65
Notes
Journal Article
Research Support, Non-U.S. Gov't --- Old month value: Dec
Résumé
Protein-bound steroids can be separated from free steroids using microcolumns of silica gel coated with an hydrophobic (octadecyl) solid phase. The bound fraction is eluted in the assay buffer, whereas the free fraction is retained quantitatively on the column in the first step and can be recovered in methanol. Both fractions can be quantitated directly (e.g. by liquid scintillation spectrometry when using radioactive ligands) or kept for further analysis (e.g. by TLC, HPLC etc.). Separation of the bound and free fractions is rapid, accurate and reproducible; intra- and inter-assay coefficients of variation are lower than 5 and 10%, respectively. Recovery of radioactive steroids is high (usually over 85%) and can be estimated separately for each sample. Since assay blanks are very low (typically less than 0.1% of input), this new method, which could be termed "hydrophobic interaction chromatography" (HIC), should prove especially useful for the development of sensitive binding assays, particularly in the field of steroid receptors. The HIC method compared well with three methods currently used for steroid binding assays, namely adsorption of unbound steroids on dextran-coated charcoal, gel filtration on Sephadex LH-20 and adsorption of steroid-protein complexes on DEAE-cellulose filters. Examples of application described here include studies on human plasma sex hormone binding globulin (SHBG) and SP2 placental protein (saturation analysis, binding specificity etc.), the separation of antibody-bound steroids in a radioimmunoassay and the estimation of androgen binding to rat epididymal androgen binding protein (rABP). Receptor assays are illustrated by saturation analysis of the mouse uterine oestrogen receptor and of the androgen receptor in the human genital skin.
Mots-clé
Androgen-Binding Protein/analysis Animals Carrier Proteins/*analysis Chromatography/methods Epididymis/metabolism Female Humans Male Mice Placenta/metabolism Pregnancy Proteins/analysis Protein Binding Rats Receptors, Androgen/analysis Receptors, Estrogen/analysis/isolation & purification Receptors, Steroid/*analysis Sex Hormone-Binding Globulin/analysis Steroids/*isolation & purification Uterus/metabolism
Pubmed
Web of science
Création de la notice
25/01/2008 10:31
Dernière modification de la notice
20/08/2019 16:10
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