Blue native PAGE

Protein-ligand or protein-protein interaction regulates various biological processes. Because these interactions are often mediated by noncovalent bond, the detection of the complexes should be carried out under the conditions where the complexes remain intact. Gel filtration chromatography has been historically used for the analysis of such complexes. However, this method has some drawbacks: a low throughput, dilution effects and requiring the large volumes of buffer and relatively large amounts of specimens. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an alternative way to analyze macromolecular complexes. It is relatively high throughput and ideal for the downstream experiments, such as multi-dimensional electrophoresis, isolation of the complexes and Western blot. In BN-PAGE, the samples are mixed with a negatively charged dye Coomassie Brilliant blue G-250 (not R-250) for the crude ones or Ponceau S for the purified complex, which binds to proteins and allows for the separation of the complexes according to their molecular sizes during the electrophoresis