Microassay for proteins on nitrocellulose filter using protein dye-staining procedure.

A simple and rapid microassay for proteins utilizing the protein dye-staining procedure with a nitrocellulose (NC) filter is described. Proteins were directly bound to an NC filter using the "BIO DOT" microfiltration apparatus to ensure their uniformity. The proteins were then stained with a dye (Ponceau Red 3R or amido black 10B), and the optical density of the stained protein spots was directly measured by a densitometer. A good linearity between the optical density and the amount of protein was obtained in the range 0.05 to 10 micrograms. A larger number of samples (up to 96 samples) could be assayed within 1.5 h simultaneously. Contaminating chemicals, such as amino acids, sugars, reducing agents, chelating agents, tris(hydroxymethyl)aminomethane, deoxyribonucleic acid, and nucleotides, did not interfere with the assay. The reproducibility, pH dependency, and application of the assay to the quantitation of a small amount of proteins in body fluids are discussed.