Paul Menter, Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547 USA
The unparalleled resolution and flexibility possible with polyacrylamide gel electrophoresis (PAGE) has led to its widespread use for the separation of proteins and nucleic acids. Gel porosity can be varied over a wide range to meet specific separation requirements. Electrophoresis gels and buffers can be chosen to provide separation on the basis of charge, size, or a combination of charge and size.
The key to mastering this powerful technique lies in the polymerization process itself. By understanding the important parameters, and following a few simple guidelines, the novice can become proficient and the experienced user can optimize separations even further.
This bulletin takes a practical approach to the preparation of polyacrylamide
gels. Its purpose is to provide the information required to achieve reproducible,
controllable polymerization. For those users interested only in the bare
essentials, the Polymerization Protocols can be used as a laboratory
Mechanism of Polymerization
Polyacrylamide gels are formed by copolymerization of acrylamide and bis-acrylamide (bis, N,N'-methylene-bisacrylamide). The reaction is a vinyl addition polymerization initiated by a free radical-generating system (Chrambach 1985). Polymerization is initiated by ammonium persulfate and TEMED (tetramethylethylenediamine): TEMED accelerates the rate of formation of free radicals from persulfate and these in turn catalyze polymerization. The persulfate free radicals convert acrylamide monomers to free radicals which react with unactivated monomers