Figure 1. Isoelectric focusing – the first dimension in 2DE. Sample introduction by rehydrating the immobilized pH gradient (IPG) strip in the protein sample results in a distribution of protein along the entire length of the strip. A protein of pI 5.5 has different charge states at various positions along the pH gradient. After a sufficiently long isoelectric focusing (IEF) step, proteins in the IPG reach their equilibrium positions at their respective pIs.

Figure 2. Zooming in on target proteins with broad- and narrow-range immobilized pH gradients (IPGs) in 2DE. A broad-range IPG and a gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) is used to get an overview of the 2DE map and to survey for proteins of interest. A more focused analysis with overlapping narrow-range IPGs spanning a pI range from 3 to 6.5 can improve resolution of proteins. Choices of IPG, pH range and density of SDS-PAGE gels can be used to isolate a region of interest. The numbers cited illustrate the greater number of proteins visualized in a targeted approach. The full and shaded ovals are landmarks, representing the same proteins in the various gels.

Figure 3. Identifying differentially expressed proteins with 2DE mass spectrometry (2DE-MS). The workflow for a 2DE-MS approach to studying differential protein expression in cells with or without drug treatment is illustrated here. Proteins from an equivalent number of cells are extracted and solubilized in a buffer suitable for 2DE. After 2DE separation, proteins are stained and visualized with a protein stain. Comparison of gel images is performed and spots that are found differentially expressed (spots marked by arrows) are excised. Proteins are digested in the gel and peptides extracted and identified following matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS analyses. The combination of mass peaks in the mass spectrum identifies a protein in peptide mass fingerprinting (MW: molecular weight; m/z: mass-to-charge ratio).