A modified bromosulfalein assay for the quantitative estimation of protein is described that is simple, rapid, and sensitive. The assay can be used in the range between 0.5 and 100 ~g of protein with samples containing from about 0.01 to 5 mg of protein/ml. Estimates of protein levels in,homogenates Of various tissues obtained by the bromosulfalein assay compared welI with those obtained by the more familiar Lowry method.
A number of methods have been devised to estimate the concentration of proteins in dilute solutions (l-6). Each of these methods has its advantages and disadvantages. In general, the more sensitive methods are complicated and time consuming, while the simpler methods are less sensitive.
Greif (7) first described the use of the anionic dye bromosulfalein to estimate protein concentration.
This dye binds rather specifically to proteins (probably via interaction with cationic groups) and, in acid solution, the protein/dye complex precipitates. The basis of Greif's assay was to separate bound from unbound dye by centrifugation, to resuspend the precipitate in base, and then to measure the amount of bound dye spectrophotometrically.
As originally described, this method was rather cumbersome to carry out and required on the order of 100 pg of protein. This assay was later modified by Nayyar and Glick (8) and then Bonting and Jones (9) to increase its sensitivity (1 -10 pug of protein) and speed. In these methods, the amount of protein is determined by measuring the amount of dye which remains in solution after acid precipitation of the protein/dye complex. In carrying out this modified assay, we have noticed several limitations.
First, the range is rather limited (between 1 and 10 yg of protein). Second, when small amounts of protein (~3 pg) are assayed, the absorbance of the final supernatant is only slightly different from the blank (no protein) value, resulting in the familiar problem of measuring a small difference between large numbers. Finally, 75