Preparation of protein samples for SDS-polyacrylamide gel electrophoresis: procedures and tips

odium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) is the most widely used analytical method to resolve separate components of a protein mixture. It is almost obligatory to assess the purity of a protein through an electrophoretic method. SDS-PAGE simultaneously exploits differences in molecular size to resolve proteins differing by as little as 1% in their electrophoretic mobility through the gel matrix (1). The technique is also a powerful tool for estimating the molecular weights of proteins (2, 3). The success of SDS-PAGE as an indispensable tool in protein analysis has been attributed to three innovations that permitted the correlation of electrophoretic mobility with a protein’s molecular mass (4). First was the introduction of discontinuous buffer systems where the sample and gel running buffers differ in both composition, TrisHCl/Tris-glycine, and pH, 6.8/8.3, respectively (5, 6). Discontinuous buffer systems allow larger sample volumes to be loaded while maintaining good resolution of sample components because the proteins are focused, or “stacked,” as thin bands prior to entering the resolving gel. Second was the use of the detergent sodium dodecyl sulfate (SDS) and reducing agents to denature proteins (7). SDS binds strongly to proteins at an approximate ratio of 1 dodecyl sulfate molecule per 2 amino acid residues (8). Therefore, the negative charge/unit mass ratio when SDS is bound to the polypeptide chain is similar for all proteins. Third was the combination of the first two discoveries employing a simple Tris-glycine buffer system (9). More recently, buffer combinations such as Tris-borate (10) and Tristricine (11) have improved the resolving power of the original methods. Modern SDS-PAGE has evolved to use microslab precast gels (12). Precast and packaged gels in a wide variety of gel formulations, acrylamide percentages, thicknesses, well formats, and buffer systems are now commercially available from several manufacturers. Therefore, successful SDS-PAGE analysis of protein samples no longer depends on tedious gel casting, buffer preparation and apparatus set-up, but on careful sample preparation and treatment prior to loading the gel. This article describes techniques and procedures as a guide for preparation of protein samples for SDS-PAGE analysis.