Detection of Proteins and Sialoglycoproteins in Polyacrylamide Gels Using Eosin Y Stain

Abstract

A rapid, sensitive, and reliable staining technique is essential in detection of proteins in polyacrylamide gels. Coomassie brilliant blue R-250 (CBB) is the stain that meets these criteria except for sensitivity; i.e., CBB staining requires relatively large amounts of proteins. It has been reported that the sensitivity for CBB stain in polyacrylamide gels is 0.1–0.5 μg/protein band (1). This problem of relatively low staining sensitivity is often circumvented by employing silver staining techniques (2–5). However, it is difficult to transfer silver stained proteins to transfer membranes unless they either are negatively stained by silver (6) or the positively silver-stained proteins are treated with 2X SDS sample buffer prior to transfer (7). In addition, sialoglycoproteins cannot be detected by CBB and thus have to be visualized by other stains, such as the periodic acid-Schiff (PAS) reagent (8), silver stains (9), or silver/Coomassie blue R-250 double staining technique (10).

To circumvent the deficiencies of the above staining techniques, we have developed an eosin Y staining technique (11). This staining method allows one to detect proteins more rapidly than most CBB and silver staining methods. It detects a variety of proteins in amounts as little as 10 ng in polyacrylamide gels, including membrane sialoglycoproteins, and has the added advantage of the antigenicity of the stained proteins being retained. The precise mechanism by which eosin Y stains both proteins and sialoglycoproteins is not fully understood.