Immunoprecipitation and characterization of membrane protein complexes from yeast

In this undergraduate biochemistry laboratory experiment, the vacuolar ATPase protein complex is purified from yeast cell extracts by doing immunoprecipitations under nondenaturing conditions. Immunoprecipitations are performed using monoclonal antibodies to facilitate data interpretation, and subunits are separated on the basis of their molecular size by SDS-PAGE. Both integral membrane and peripheral subunits are detected in Coomassie Blue-stained gels, and the identity of several subunits are confirmed by Western blots. Western blots serve to illustrate reproducibility and authenticity of the results and help students understand that they could use immunoblotting to detect subunits that are not visualized by Coomassie stain. The molecular mass of the subunits is estimated by their migration relative to molecular marker proteins in the same gel and used to estimate the size of the entire complex. Just like in biochemistry research, students learn to integrate several techniques to solve the subunit composition of a protein complex. These experiments are an alternative to classical protein purification techniques and can be adapted to study most protein complexes composed of membrane-bound and peripheral subunits from yeast. Instructors can use this approach to teaching proteomics and introduce students to systems biology. Because numerous yeast proteins are epitope tagged, instructors could use commercially available antibodies to study a protein complex of interest.