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Blue native PAGE

Abstract

Blue native PAGE (BN-PAGE) can be used for one-step isolation of protein complexes from biological membranes and total cell and tissue homogenates. It can also be used to determine native protein masses and oligomeric states and to identify physiological protein–protein interactions. Native complexes are recovered from gels by electroelution or diffusion and are used for 2D crystallization and electron microscopy or analyzed by in-gel activity assays or by native electroblotting and immunodetection. In this protocol, we describe methodology to perform BN-PAGE followed by (i) native extraction or native electroblotting of separated proteins, or (ii) a second dimension of tricine-SDS-PAGE or modified BN-PAGE, or (iii) a second dimension of isoelectric focusing (IEF) followed by a third dimension of tricine-SDS-PAGE for the separation of subunits of complexes. These protocols for 2D and 3D PAGE can be completed in 2 and 3 days.

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Figure 1: Separation of dodecylmaltoside-solubilized mitochondrial complexes exemplifying the native mass range (<100 kDa to 10 MDa) covered by BN-PAGE.
Figure 2: Separation of supramolecular assemblies of oxidative phosphorylation complexes by 1D BN-PAGE and identification of their constituent individual complexes by 2D BN/BN-PAGE.
Figure 3: Analysis of subunits of the mitochondrial ATP synthase complex from Arabidopsis by 3D BN/IEF/SDS-PAGE.

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Sonderforschungsbereich 628, Project P13 to H.S. and DFG grant Br1829-7/2 to H.P.B.

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Correspondence to Hermann Schägger.

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Wittig, I., Braun, HP. & Schägger, H. Blue native PAGE. Nat Protoc 1, 418–428 (2006). https://doi.org/10.1038/nprot.2006.62

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