One- and two-dimensional blue native-PAGE and immunodetection of low-abundance chloroplast membrane protein complexes.

ABSTRACT

Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful method for separating protein complexes from biological membranes under native conditions. BN-PAGE provides much higher resolution than gel filtration or sucrose density gradient centrifugation, and it can be used to estimate the molecular mass of protein complexes. First, membrane protein complexes need to be solubilized with a mild nonionic detergent such as digitonin or dodecyl maltoside. Coomassie brilliant blue G-250, a negatively charged dye that binds to the surface of the solubilized complexes, is then added so these can be resolved according to their size by non-denaturing (native) electrophoresis. BN-PAGE can be combined with a second dimension SDS-PAGE step (two-dimensional (2D)-BN/SDS-PAGE), so that the subunits making up these complexes are also separated according to their size. Here, we present our 2D-BN/SDS-PAGE method, and subsequent immunoblotting method, for the detection of relatively low-abundance proteins from plant chloroplasts.