I come back to my blog today to present you another approach of the electrophoresis : the Blue Native-PAGE.
Before being able to find this technique, I was looking for a way to visualize the complex that I am studying during my PhD : the stressosome. Because this complex is anchored to the bacterial membrane in Listeria monocytogenes, first I was looking for how protein complexes have ever been studied and I had to deal, unfortunately, with the incompatibility of membrane proteins with commonly used protein interaction assays…. However, an attractive solution has been found during the 90’s to resolve respiratory complexes in mitochondria : the Blue Native-PAGE !!! I have decided to show you an overview because it totally differs from classical SDS-PAGE (I would like to thank Lara Kricks who works in the same department of microbiology to have taken the time to show me the protocol).
The first and the most important thing is to do everything at 4ºC !! Consequently, you spend a lot of time in the cold room and you have to get dressed like in winter whereas it is almost 45ºC in Madrid !! Fortunately, coats are available.
Blue native electrophoresis is a charge shift method : the anionic dye Coomassie G-250 binds to proteins because of its hydrophobic properties. The binding of dye molecules imposes a charge shift on the proteins that are then separated according to size in acrylamide gradient gels. Because negative charges (from Coomassie G-250) repeal each other, it allows to reduce the tendency of membrane proteins to aggregate. Moreover, the most advantageous is that upon binding of the dye, membrane proteins convert into water soluble proteins : detergents are not required and we maintain proteins in their native state without any protein denaturation.
After the electrophoresis, I follow with a classical Western-Blot. At the beginning, it is surprising to work with a blue membrane !!!
Thanks to this technique, I would be able to determine protein masses of the complex by using antibodies against the proteins of the stressosome. Moreover, Blue Native-PAGE would allow me to study the oligomeric states and to identify protein-protein interactions !!! I hope it will work, I have just started !!!