Electrophoresis

The 10X Tris-Glycine Buffer, with a pH of 8.3, is designed specifically for use in native PAGE gel electrophoresis. This buffer offers great convenience, as it can be diluted with distilled water or another suitable solvent according to the specific application prior to electrophoresis. Additionally, it is useful for the transfer of protein samples onto nitrocellulose membranes during western blotting. To prevent the gel from swelling during the transfer process and to enhance the binding of protein samples to nitrocellulose, the inclusion of methanol is essential.

SDS-PAGE is a method employed for separating proteins via electrophoresis, based on the principle that charged molecules migrate through a matrix in response to an applied electric field. The matrix utilized for this separation is polyacrylamide, which is derived from acrylamide, a compound that can be hazardous. Polyacrylamide is commonly used for the size-based separation of proteins and nucleic acids. The gel matrix forms through the free radical polymerization of acrylamide along with a crosslinking agent known as N, N-Methylenebisacrylamide. In this process, acrylamide monomers polymerize into long chains, initiated by a free radical-generating system, and these chains are linked by the cross-linker, resulting in a gel structure. This gel is crucial for effective electrophoretic separation, facilitating the analysis of biomolecules based on size.