S odium dodecyl sulfate polyacry- lamide gel electrophoresis (SDS- PAGE) is the most widely used ana- lytical 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 differ- ences in molecular size to resolve proteins differing by as little as 1% in their elec- trophoretic 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 in- dispensable tool in protein analysis has been attributed to three innovations that permit- ted the correlation of electrophoretic mobil- ity 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, Tris- HCl/Tris-glycine, and pH, 6.8/8.3, respec- tively (5, 6). Discontinuous buffer systems allow larger sample volumes to be loaded while maintaining good resolution of sample components because the proteins are fo- cused, 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 pro- teins (7). SDS binds strongly to proteins at an approximate ratio of 1 dodecyl sulfate molecule per 2 amino acid residues (8). T herefore, the negative charge/unit mass ratio when SDS is bound to the polypeptide chain is similar for all proteins. T hird was the combination of the first two discoveries employing a simple Tris-glycine buffer system (9). More recently, buffer combina- tions such as Tris-borate (10) and Tris- tricine (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, acry- lamide percentages, thicknesses, well for- mats, and buffer systems are now commer- cially available from several manufacturers. T herefore, successful SD S-PAGE analysis of protein samples no longer depends on te- dious gel casting, buffer preparation and ap- paratus set-up, but on careful sample prepa- ration and treatment prior to loading the gel. This article describes techniques and procedures as a guide for preparation of pro- tein samples for SDS-PAGE analysis. Sample buffer preparation To ensure consistent and successful PAGE analysis, the highest purity reagents should be used to prepare sample buffer stock solutions. After a reliable source of electrophoresis reagents has been identified, the vendor and buffer component chemicals should be maintained. High purity elec- trophoresis, Ultrol ® grade, and molecular bi- ology grade reagents are available through Novagen’s partner brand, Calbiochem. Solutions must be carefully and safely pre- pared, dated, and chemical lot numbers recorded. Concentrated stock solutions should not be stored for long periods of time. Tris base, rather than Tris-Cl, should be used for buffer preparation and pH ad- justment made with HCl. Use of Tris-Cl 10 inNovations 13 Preparation of protein samples for SDS-polyacrylamide gel electrophoresis: procedures and tips Anthony C. Grabski 1 and Richard R. Burgess 2 — 1 Novagen, Inc. and 2 McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI 53706