A Coomassie blue-binding assay for the microquantitation of immobilized proteins

A sensitive assay procedure for the determination of microgram quantities of immobilized proteins is described. The procedure is based on the property of Coomassie blue G-250 to bind strongly yet reversibly to proteins. The assay involves incubation of the immobilized protein with a solution containing 0.1% Coomassie blue, 10% acetic acid, and 25% isopropyl alcohol in distilled water at room temperature followed by washing off of the unbound dye. The protein-bound dye is eluted with methanolic NaOH, acidified, and the absorbance is measured at 605 nm. The assay is highly reproducible and several proteins immobilized on various matrices could be conveniently assayed. Protein values determined by the dye-binding assay showed good agreement with those obtained by other procedures.