A miniature molecular-sieving column assay for cytoplasmic vitamin A-binding proteins.
Cytoplasmic vitamin A-binding proteins were measured by a method using centrifugation of gel-exclusion columns and compared to the sucrose gradient method. The gel-exclusion method analyzed 18 samples simultaneously on one table-top centrifuge, while the sucrose gradient method required use of three ultracentrifuges to process 18 samples simultaneously. Multiple 2-min low-speed centrifugations of test cytosol applied to miniature molecular-sieving columns was a faster (1/2 the working time for 18 samples), more convenient, and more accurate method for measuring cytoplasmic vitamin A-binding proteins than was the sucrose gradient method. Using the same cytosol and [3H]retinoid preparations, the rapid gel-exclusion method results were only slightly lower than those obtained by radioimmunoassay but 50% higher than the values obtained by the sucrose gradient assay. The methodology described may be useful not only for cytoplasmic vitamin A-binding proteins but also for other similar binding protein assays.
D000818:Animals; D001667:Binding, Competitive; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel / Q000379:methods; D003593:Cytoplasm / Q000032:analysis*; D003600:Cytosol / Q000032:analysis; D008297:Male; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains; D012177:Retinol-Binding Proteins / Q000032:analysis*; D012588:Scintillation Counting; D013737:Testis / Q000032:analysis
D012177:Retinol-Binding Proteins