Protein determination by ponceau S using digital color image analysis of protein spots on nitrocellulose membranes

A procedure was developed to estimate protein concentrations using color im age analysis of protein spots stained with ponceau S. The method involved s potting a constant volume (2 mu l) of the protein solutions on nitrocellulo se paper, staining with acidic ponceau S, destaining, and air drying the pa per. The image of the nitrocellulose paper was grabbed using a digital colo r scanner and thresholded with an optimal value to mark the area of the spo t. The intensity of the color in the spot was measured in an arbitrary unit of intensity termed as inverse integrated gray value. This value showed a discernible increase with protein concentrations from 0.1 to 50 mu g protei n per spot (0.05-25 mg/ml). The method is simple and convenient compared to the conventional spectrophotometric procedures and allows several samples to be analyzed simultaneously. It can also be used to estimate protein conc entration in the spots stained with Coomassie brilliant blue or Other dyes. (C) 1999 Academic Press.