Vertical Agarose Electrophoresis and Electroblotting of High-Molecular-Weight Proteins

Abstract

AbstractVery large molecular weight proteins are difficult to separate by electrophoresis because of their poor penetration into gels using the Laemmli SDS polyacryla-mide system (1). Protein migration in SDS gels has been found to be linear with the log of the molecular weight (2), so the larger the protein, the more poorly it is resolved from other big proteins. Many workers have attempted to solve this problem by using very low concentration acrylamide gels (3), acrylamide mixed with agarose (4), or acrylamide gradients (5) to better separate large proteins. Low concentration acrylamide gels are difficult to use because of their mechanical fragility and distortion during handling; these problems are magnified when blotting is attempted. An additional difficulty in blotting very large proteins is their poor transfer to the membrane. Inclusion of 2-mercaptoethanol in the transfer buffer improves transfer efficiency, but acrylamide gels stained after transfer typically still contain most of the giant muscle protein titin (6).A new electrophoresis system using SDS agarose for protein electrophoresis and blotting has been described (7). An example showing the resolution for several muscle samples containing large proteins is shown in Fig. 1. Migration distance shows a linear relationship with the log of the molecular weight (7). This system allows quantitative transfer of proteins from the gel with much higher reproduc-ibility than can be achieved with methods using low percentage acrylamide.KeywordsAmmonium PersulfateTransfer BufferHigh Molecular Weight ProteinSaran WrapAcrylamide GradientThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.