Recently we reported a highly sensitive fluorometric assay for proteins using fluorescamine
(1). Two procedures were described. In the first method, fluorescamine was added to a buffered protein solution, and the fluorescence was measured in the reaction tube. A drawback of this procedure has been the requirement for protein samples to be free of lowmolcular-weight primary amines, since these substances interfere. The second method involved the continuous monitoring of a gel filtration column, which resolved protein from low-molecular-weight contaminants. However, the latter method was not suitable for multiple assays since a single analysis took too much time. Also, it involved more elaborate equipment and, therefore, was costly. Now we wish to report a simple, rapid procedure which includes the separation of proteins from lowmolecular-weight, material prior to assay. Separation was achieved by gel filtration through columns prepared as follows:
1.20 g Sephadex G-25 fine (Pharmacia, Piscataway, NJ') was swollen in 0.05 M phosphate buffer, pH 8, containing 1% Dowidcide as mold inhibitor (Harleco, Philadelphia, PA). The gel suspension was then quantitatively transferred into a 20 X 0.7 cm disposable glass column (Bio-Rad, Richmond, CA)! in which the original bed support had been replaced by a sturdier porous polyethylene disc. Discs were punched from l-mm-thick sheets (Bel-Art Prod., Pequannonk, NJ). Enough buffer was allowed to flow through the column for complete settling of the gel bed (column height of 16 cm). The bed was then covered with another polyethylene disc of 4-mm thickness. This disc efficiently prevented the column from running dry over a period of several hours (presumably by retaining liquid due to capillary forces) and also facilitated the introduction of sample and buffer onto the column. For the processing of a large number of samples, as many as 20 columns were run simultaneously. 559