High resolution two-dimensional electrophoresis of native proteins.

Blue native PAGE (BN-PAGE) is a powerful method to separate protein complexes while preserving their native state. However, the resolution of the method is limited as complexes with similar molecular masses cannot be resolved. Here we describe native 2DE using immobilized pH-gradients in combination with BN-PAGE to resolve protein complexes by their pI and molecular mass. This method enables electrophoretic separation of proteins between pI 3 and 10 and can resolve molecular masses up to 1.2 MDa. Visualized gel spots at large molecular weight were identified using MS to confirm potential protein complexes. Several protein complexes could be identified, most prominent GroEL in complex with GroES, parts of the ribosomal machinery and membrane transport system. In summary, this method enables easy high-resolution electrophoretic separation of protein complexes.