This paper reports both the experimental application and two-dimensional simulation of nonlinear electrophoresis (ITP/IEF) of proteins in a microfluidic chip. A two-dimensional ITP model has been developed from a one-dimensional model and simulated using the Nernst-Planck equations. The concentration stacking and separation features of ITP are explored by simulations of three virtual proteins. Experiments of ITP demonstrated that a mixture of three fluorescent proteins were concentrated and stacked into three adjacent protein zones under a constant voltage of 100 V over a 2 cm long microchannel. The self-sharpening behavior of ITP zones dispersed by a T-junction was clearly demonstrated both by experiments and simulations. Two dimensional simulation of isoelectric focusing of proteins has been first developed by using existing solvers in Femlab. 8 carrier ampholytes which have pIs from pH 3 to pH 10 were employed in this simulation to generate a pH gradient of 3-10 in a 300micron x 2cm microchannel. Each ampholyte has three charge states of -1, 0 and 1 which are simulated as three individual components, but related to each other by finite reactions. A virtual protein with 7 charge states was simulated to demonstrate the characteristics of isoelectric focusing of proteins. Simulation revealed that increase of current density results in a stepwise pH gradient from pH 3 to pH 10 and more ampholytes would generate more linear pH gradient. The simulation also demonstrated that the protein is being focused via the well-known double-peak approach to its pI position. Experiments of IEF demonstrated that several fluorescent proteins were focused in a 2 cm long microchannel in 3-10 min using broad-range ampholytes at electric field strengths ranging from 25 to 100 V/cm.