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.