Premium
Focused proteomics: Monoclonal antibody‐based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stain
Author(s) -
Murray James,
Marusich Michael F.,
Capaldi Roderick A.,
Aggeler Robert
Publication year - 2004
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200406006
Subject(s) - phosphoprotein , phosphorylation , monoclonal antibody , biochemistry , cytochrome c oxidase , oxidative phosphorylation , microbiology and biotechnology , dephosphorylation , chemistry , protein phosphorylation , biology , mitochondrion , phosphatase , antibody , protein kinase a , immunology
We have raised monoclonal antibodies capable of immunocapturing all five complexes involved in oxidative phosphorylation for evaluating their post‐translational modifications. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F 1 F 0 ATP synthase) from bovine heart mitochondria were obtained in good yield from small amounts of tissue in more than 90% purity in one step. The composition and purity of the complexes was evaluated by Western blotting using monoclonal antibodies against individual subunits of the five complexes. In this first study, the phosphorylation state of the proteins without inducing phosphorylation or dephosphorylation was identified by using the novel Pro‐Q Diamond phosphoprotein gel stain. The major phosphorylated components were the same as described before in sucrose gradient enriched complexes. In addition a few additional potential phosphoproteins were observed. Since the described monoclonal antibodies show cross reactivity to human proteins, this procedure will be a fast and efficient way of studying post‐translational modifications in control and patient samples using only small amounts of tissue.