Obtaining replicas from a polyacrylamide gel at different stages of the same electrophoretic run

Abstract

Varying the duration of electrophoresis produces different patterns in the same gel at the same power settings. However, with the present methods for visualizing proteins, only one electrophoretic separation can be obtained from one electrophoretic experiment. Thus, only a part of each electrophoretic experiment’s analytical potential is usually exploited. This is particularly evident in isoelectric focusing (IEF), where, during one experiment, nonequilibrium isoelectric focusing (NEPEGE) is followed by true equilibrium focusing, and each phase makes it possible to obtain very different separations (see ref. 1 for a review). The transfer of proteins from a gel to a membrane by simple contact (print technique) ha8 been described (2). However, in this procedure th membrane is impregnated with a substrate-containing solution that passes through the gel and does not allow further electrophoretic separation to proceed. To our knowledge, the transfer of proteins by contact with a clean membrane such that the functional character of the gel is retained has not been reported, despite the simplicity, ease, and possible applications of this procedure. We recently found that some erythrocyte proteins can be transferred from a polyacrylamide gel to a clean cellulose acetate membrane by simple contact. The replica obtained can be stained with a convenient solution, making the proteins visible on the membrane. The most important and innovative feature is that this simple procedure does not impair the functioning of the gel, thus allowing additional replicas to be obtained from a gel at different stages of the same electrophoretic run. Here we report an IEF method for the sequential visualization of the polymorphisms of acid phosphatase (ACP1; EC 3.1.3.1), esterase D (EsD; EC 3.1.1.1), hemoglobin beta chain (Hbp), and phosphoglucomutase (PGM1; EC 3.2.2.2) in erythrocytes. The gels [containing Ampholine (LKB, Stockholm, Sweden), pH 5-7, 20 milL, and pH 6-8, 10 milL] and the erythrocyte samples were prepared as previously described (see ref. 3, and Table 1). Cellulose acetate strips (cat. no. 01A36-100; Cellogel Chemetron, Milan, Italy) were soaked for 1 h in doubly Hb 30 PGM1 90