Blue Native Polyacrylamide Gel Electrophoresis BNPAGE

Various techniques have been developed to reveal the composition of complexes, including BN-PAGE. BN-PAGE enables protein complexes to be separated under native conditions, and then the individual proteins from a particular complex are resolved under denaturing conditions. This technique was developed to examine sol-ubilized membrane proteins from various tissues (mammalian muscle, yeast, and bacteria) and allowed separation of all the protein complexes of the oxidative phosphorylation system within one gel [61]. Their method was, until the publication of reference 62, not applicable for separating complexes from more complex mixtures such as whole-cell extracts. A simple dialysis step opened up the mammalian cell proteome to BN-PAGE separation and subsequent proteomic investigations.

BN-PAGE works by gently solubilizing cells to give a mixture of intact protein complexes, which can be separated on a non-denaturing gradient gel according to their

MW. The negative charge of the blue dye and the size and shape of each complex determines how far that complex migrates into the gel. The gel lane is then cut out and separated on a second gel orientated perpendicular to the first axis of separation and includes SDS to separate the component proteins of each complex according to their MW. BN-PAGE studies so far in plants have been restricted to membrane proteins [reviewed in reference 63]. Drykova et al. [64] used several techniques including BN-PAGE, to confirm that y-tubulin is associated with membranes in the form of large (>1 MDa) protein complexes. Respiratory enzyme complexes in mitochondria and protein-import complexes in chloroplasts have been studied in Arabidopsis in references 63 and 65, respectively.

However, not all protein complexes appear to be suitably solubilized by any one detergent, and BN-PAGE does not resolve small protein complexes (<100 kDa) well due to the small separation distance of the first gel step. Distinct complexes of similar molecular masses may co-migrate, and the constitutive proteins then appear to be present in the same complex.

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