Glycerol gradient fractionation and immunoprecipitation of F₀F₁-ATP synthase complex from T. brucei mitochondria.

(A) Western blot and native dot blot analyses of the 10–30% glycerol gradient-fractionated cleared mitochondrial lysate were performed using polyclonal antibodies against subunit b and β, and monoclonal antibody mAb64 to determine the sedimentation pattern of the F₀F₁-ATP synthase complex. MAb64 was further used to immunoprecipitate (IP) complexes from the 10S and 40S peaks and their protein compositions were analyzed by liquid-chromatography tandem mass spectrometry (LC-MS/MS). (B) Immunoprecipitated 10S and 40S complexes were fractionated on a 12% SDS PAGE gel and stained by Sypro Ruby. Protein bands corresponding to immunoglobulin heavy (hc) and light (lc) chains as well as predicted positions of F₁ subunits α, β, γ and δ and the size standards are indicated.