Quantification of Coomassie Blue Stained Proteins in Polyacrylamide Gels Based on Analyses of Eluted Dye1
Quantification of Coomassie Brilliant Blue R-250 stained proteins on cellulose acetate strips was made over a limited range of protein concentrations (1); this work extended to quantification of Coomassie Blue stained proteins on polyacrylamide gels (2). Fishbein (3) showed that the major sources of error in quantitative densitometry were protein variations in dye binding, inaccuracy in integrating small areas, and the limited range over which detection of dye was proportional to protein present. Quantification of protein present, based on amino acid composition, was carried out on polyacrylamide gel bands where total nitrogen was based on fluorescamine detection (4). In other studies proteins stained with dibromotrisulphofluorescein (5) allowed for quantification of high protein concentrations, but did not afford a simultaneous comparison of proteins present in low concentrations.
With fluorometric scanning of fluorescamine-labeled proteins rapid quantification could be obtained (6), however data again obeyed Beer's Law only over a limited range of protein concentrations, as observed in other densitometric scanning analyses.
This work was undertaken to quantitatively estimate the amount of protein present, based on total nitrogen (7), of specific polypeptides in stained bands of polyacrylamide gels, where there was a large variance in molecular weights of proteins and hence large differences in protein concentrations, as found in the heavy and light subunits of myosin (8-l 1). It was not possible to estimate concentrations of both myosin heavy chains and myosin light chain CZ of canine cardiac left ventricle from densitometric tracings of a single gel; both were not simultaneously within the range of linearity. The restricted range for determining protein concentrations [ 1-10 Fg ( 12)] was defined by the limited range over which Coomassie Blue obeyed Beer's Law. Various errors in densitometric tracings were introduced when proteins were analyzed on separate gels (1,4): varying band widths in a single percent gel introduced large errors. In the procedure presented here dye was extracted from stained bands of polyacrylamide gels with varying volumes of a solvent such as 25% dioxane or 25% pyridine in water (v/v).