Comparison of Reflectance and Transmission Densitometry, Using Document and Laser Scanners, for Quantitation of Stained Western Blots☆

Abstract The quantitation of stained Western blots by reflectance and transmission scanning was explored using a blot of monoclonal immunoglobulin G probed with anti-mouse antibody. The so-called “scanned absorbance” output from a document scanner was found to be directly proportional to the fraction of light absorbed rather than obeying the logarithmic relationship expected for true spectrophotometric absorbance. This explains observations in the literature of a strongly curved relation between loading and “absorbance.” Laser transmission densitometry of a blot immersed in a clarifying solvent mixture showed that peak area was linearly related to loading over a wide range. In reflectance mode the document scanner also gave equally linear quantitation of dry blots, providing that a logarithmic correction curve was applied during scanning. It was found advantageous to interpose a red acetate filter sheet between the blot and the scanning table to aid detection of weakly stained bands. The document scanner gave less satisfactory results when used in transmission mode on a clarified blot because weak bands were poorly quantitated.