This manuscript describes protocols for separation of complex protein samples using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Electrophoresis in a single dimension, e.g., 1D SDS polyacrylamide gels, has the potential to rapidly separate hundreds of proteins. When two orthogonal high-resolution electrophoretic methods are efficiently combined in perpendicular dimensions, complex protein mixtures can be separated into thousands of discrete spots. The most common 2D gel separation for intact proteins involves a first-dimensional separation using isoelectric focusing (IEF) followed by separation based on protein size (SDS-PAGE). Currently, most 2D gel studies rely on the use of commercially available immobilized pH gradient (IPG) gels, which provide improved ease of use and reproducibility compared with older methods. IPG gels are available in a range of sizes and different pH ranges. Resolution typically increases as the 2D gel size increases; however, difficulty of use increases sharply and throughput decreases as gel size increases. © 2019 by John Wiley & Sons, Inc.