Detection of proteins in polyacrylamide gels using an ultrasensitive silver staining technique

Polyacrylamide gel electrophoresis is a versatile and powerful tool for the analysis of biological samples and is capable of good separation and high resolution of complex protein mixtures. Although Coomassie Brilliant Blue R250 has proved ideal as a general protein stain for the more traditional applications of this method, current trends toward thinner gels, decreased sample loading (to improve resolution), and the recent developments of two-dimensional gel electrophoresis and peptide mapping techniques have necessitated increasingly sensitive detection methods. Electrophoretic separation of radioactively labeled proteins followed by autoradiography permits the detection of trace proteins (10(-4) to 10(-5)% of total protein) in a sample (1). However, problems inherent in radioactive methods include: (a) in vitro labeling may alter physical properties of proteins, and (b) in vivo experiments require excessively large quantities of isotopes that are prohibitively expensive in animals and unethical in human clinical studies. Alternative methods such as fluorescent staining (2) and heavy metal stains (3) are of less than, or at best of equivalent sensitivity to, Coomassie Brilliant Blue R250.