A minicolumn apparatus for androgen-binding protein measurement.
An easy, rapid, and sensitive assay that permits measurements of androgen-binding protein (ABP) in tissue as well as in spent media from Sertoli cells is described; this method involves the specific binding of labeled dihydrotestosterone (DHT) to ABP. The apparatus holds 36 minicolumns loaded with a DEAE Bio-Gel matrix. A peristaltic pump is used for the free fraction elution, taking into account the extremely rapid rate of dissociation of the ABP-DHT complexes. This technique, which allows Scatchard plot analysis, has been used to measure the rates of association (5.15 X 10(5)M-1 S-1 and dissociation (21.32 X 10(-4) S-1; t 1/2 = 5.5 min): the ratio of these rate constants is in perfect agreement with equilibrium dissociation constants determined by Scatchard plot analysis (KD = 4-4.5 nM). The intraassay and interassay coefficients of variation are 5 and 8%, respectively. A good correlation (r = 0.98) is obtained with the standard method of steady-state polyacrylamide gel electrophoresis below a value of 250 micrograms cytosolic proteins/gel. This apparatus, which allows either the measurement of ABP in 12 samples (in triplicates) at a saturating concentration or the analysis of two Scatchard plots (each of 6 points), is also very useful for a rapid localization of ABP during chromatographic purification.
Laboratoire de Biochimie, GS CNRS 79, CHU Côte de Nacre, Caen, France.
P
P
Barbey
S
S
Fradin
S
S
Carreau
M A
MA
Drosdowsky
D000727:Androgen-Binding Protein / Q000032:analysis*; D000818:Animals; D002852:Chromatography, Ion Exchange / Q000295:instrumentation; D013196:Dihydrotestosterone; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D012708:Sertoli Cells / Q000032:analysis
D000727:Androgen-Binding Protein; D013196:Dihydrotestosterone