We demonstrate a two-dimensional microfluidic architecture that integrates polyacrylamide gel electrophoresis (PAGE) with immunoblotting in a fully automated format. This assay is designed to overcome performance limitations of conventional slab-gel immunoblotting, including multiple manual interventions, low-throughput transfer and blotting, and substantial consumption of reagents and sample. To unify PAGE with blotting in one device, this microfluidic approach makes use of high-resolution regional photopatterning of multiple polyacrylamide gel elements, and automated electrophoretic transport. A complete native immunoblot of free prostate specific antigen from human seminal fluid is demonstrated in less than 5 min. Further, the characterization of post-PAGE electrophoretic transfer showed high efficiency and minimal sample dispersion.