Blue native polyacrylamide gel electrophoresis (BN-PAGE) for analysis of multiprotein complexes from cellular lysates

24.02.2011

Fiala GJ, Schamel WW, Blumenthal B

J Vis Exp. 2011;(48).

J Vis Exp          online article

The formation of multiprotein complexes (MPCs) plays a crucial role in cell signalling. Therefore, studying signal trasnduction requires characterization of MPCs to gain an understanding of protein function and regulation. For the identification and analysis of MPCs, they can be separated under native conditions using Blue Native (BN)-PAGE. Indeed, BN-PAGE is used by several groups in BIOSS, but is not always easy to perform. Therefore, we made a video, that describes this technique using the journal of visualized experiments. Using BN-PAGE, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. In this publication, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is one of the crucial steps. Using first dimension BN- and second dimension SDS-PAGE, MPCs can be subdivided into their individual constituents. Visualization of the MPC components upon gel separation can be done by standard techniques as silver staining or immunoblotting. Please note the typical black forest prezels at the end of the video.