Sensitive Staining of Acidic Dentin Proteins with Electrophoresis

Dentin phosphoprotein (phosphophoryn, DPP) is a unique acidic protein in dentin. Except for type I collagen, DPP is the most abundant extracellular matrix protein in dentin. This protein is characterized by high contents of phosphoserine (45-50%) and aspartic acid (35-38%) (Butler et al., 1995)1). Because of the high levels of aspartic acid and phosphoserine, this protein has polyanionic characteristics. The isoelectric point of this protein is 1.1 (Jonsson et al., 1978)2). DPP can bind a large number of calcium ions and affect in vitro mineralization. It forms insoluble aggregates in the presence of calcium ions (Kuboki et al., 1979)3). DPP is thought to play important roles in biomineralization. A problem with the study of DPP is the difficulty in detection of this protein with high sensitivity. Because of DPP's highly acidic character, it is difficult to detect it with standard protein stainings, e.g. Coomassie Brilliant Blue or silver staining. A number of acidic proteins, such as DPP, are poorly stained on acrylamide gels using Coomassie blue or silver nitrate staining. Alcian blue or Stains-All have been used to stain acidic proteins (Sabsay et al., 1991)4), but both stainings have lower sensitivity than silver staining. Moller et al., (1993) used combined Alcian Blue and Silver staining to stain proteoglycan sensitively (H.J. Moller et al., 1993)5). A combination of Stains-All and Silver staining was also used to stain acidic proteins in bone (Goldberg et al., 1997)6). In this study, we attempted to apply combined staining for acidic dentin proteins, especially DPP. We analyzed the dentin proteins extracted from a fragment of a single rat tooth.