Native immunoblotting of blue native gels to identify conformation-specific antibodies.
A large repertoire of immunological methods permits monitoring the interaction of antibodies with their specific antigen. However, recognition of a protein by a conformation-specific antibody represents a challenge because native conditions must be kept throughout the assay. Native immunoblotting of blue native gels conserves the native state by using Tween 20 instead of methanol for the obligatory destaining of the blot membrane. We validate the new technique with a set of monoclonal antibodies against respiratory NADH:ubiquinone oxidoreductase.
Zickermann
Volker
V
Goethe-Universität, Fachbereich Medizin, Molekulare Bioenergetik, Cluster of Excellence Frankfurt, Macromolecular Complexes, Center for Membrane Proteomics, Frankfurt am Main, Germany. zickermann@zbc.kgu.de
Wumaier
Zibiernisha
Z
Wrzesniewska
Blanka
B
Hunte
Carola
C
Schägger
Hermann
H
D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000276:immunology; D004396:Coloring Agents / Q000737:chemistry*; D042967:Electron Transport Complex I / Q000032:analysis* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D015151:Immunoblotting / Q000379:methods*; D012394:Rosaniline Dyes / Q000737:chemistry*; D025062:Yarrowia / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D004396:Coloring Agents; D012394:Rosaniline Dyes; C048139:Coomassie blue; D042967:Electron Transport Complex I