Use of lectins for detection of electrophoretically separated glycoproteins transferred onto nitrocellulose sheets

Abstract

A method of rapidly identifying lectin-binding glycoproteins separated by polyacrylamide gel electrophoresis is described. The method is particularly useful for comparing the glycoprotein content of different cell types and fractions. Normal rat liver, Novikoff hepatoma, and rat mammary tumor cell line 13762 MAT-B were fractionated to give purified nuclei and other fractions defined by their sedimentation properties in low ionic strength buffer. The subcellular fractions were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, transferred to nitrocellulose sheets, and localized by an immunochemical method to identify lectin-binding activities. The localization pattern of concanavalin A and wheat germ agglutinin-binding activities in the fractions from the three cell types showed the greatest similarities between the glycoprotein contents of normal liver and Novikoff hepatoma fractions. On a per-cell basis the purified nuclei from each of the cell types contained less activity overall than did other particulate cell fractions. Washing the nuclei from normal liver and Novikoff hepatoma, but not MAT-B cells, in nonionic detergent removed or depressed most of the lectin-binding activities. However, two major bands were unaffected by the detergent. One of these localized with wheat germ agglutinin at an apparent molecular weight of 62,000 in the nuclei of all three cell types. The other localized with concanavalin A at an apparent molecular weight of 200,000 in normal liver and Novikoff hepatoma nuclei.