Glycoprotein molecular-weight estimation using sodium dodecyl sulfate-pore gradient electrophoresis: Comparison of Tris-glycine and Tris-borate-EDTA buffer systems

The accuracy of molecular-weight estimates for glycoproteins was evaluated by sodium dodecyl sulfate-pore gradient electrophoresis. The methods for the gradient gel preparation and the electrophoretic conditions are presented in detail. Three glycoproteins of known molecular weights-ovalbumin, avidin, and fetuin, with respective carbohydrate contents of 2-3, 8-10, and 22%-were evaluated in a Tris-glycine buffer system and a Tris-borate-EDTA buffer system with and without reduction by β-mercaptoethanol. A high degree of accuracy in the molecular-weight estimates for these glycoproteins was obtained using several of these systems. This suggests that both the gradient gel system as well as the formation of borate complexes with the individual carbohydrate moieties of the glycoprotein contribute to the precision of the molecular-weight estimate. Deviations in the molecular-weight estimates in the reduced and nonreduced state, however, imply that the physical properties of the individual glycoprotein ultimately govern the accuracy of the molecular-weight estimate. Based on these observations, a strategy is presented for estimating the molecular weights of glycoproteins by sodium dodecyl sulfate-pore gradient electrophoresis, particularly as related to complex membrane systems.