The Roles of Acetic Acid and Methanol During Fixing and Staining Proteins in an SDS–Polyacrylamide Electrophoresis Gel

After SDS–polyacrylamide gel electrophoresis proteins are “fixed” in the gel to prevent dispersion of the proteins and visualized by staining with a chromogenic dye. Dyes like Coomassie Blue R-250, Amido Black, and Direct Red 81 are usually dissolved in an acetic acid–methanol–water mixture. During staining the dye solvent mixture infuses the gel and interacts with the protein. Acetic acid and methanol denature the protein and provide an acidic environment enhancing the interactions with dyes. After staining, the dye that is in the gel and not bound to the protein, is removed using the solvent medium the dyes were dissolved in. Over 2–3 h the solution surrounding the gel becomes colored, the gel becomes lighter and the protein bands remain dark and the contrast against the surrounding gel improves. This chapter describes how each of the individual components in the dye solution interact with the protein resulting in a stained protein band in a clear SDS–polyacrylamide electrophoresis gel.