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In their attempts to study the protein complexes of the respiratory chain of mitochondria, Schagger and von Jagow developed a gel-based system able to separate all of the protein complexes involved in oxidative phosphorylation in their native state [9]. This provided a new method in addition to the commonly employed enzymatic assays, den-sitometric methods, or chromatographic purification methods. By mixing the sample with CBB before the gel run, they introduced a large number of negative charges into the protein complexes, overshadowing their intrinsic charges. This was the essential step for a complete and sharp separation of the mitochondrial protein complexes. The use of CBB and the native approach were eponymous for this gel system, which consequently has been termed BN-PAGE. Combined with a denaturing SDS-PAGE gel, BN-PAGE forms the first dimension of a 2D system which is able to resolve protein complex subunits according to their affiliation to a certain complex and according to their molecular mass. The typical protein pattern on such a 2D BN/SDS-PAGE gel consists of a sample specific array of vertical rows of proteins, each row representing the subunits of a single type of protein complex (Figure 38.1). Rows on one end of the gel represent protein complexes with an HMW, while those found on the other end belong to complexes with an LMW. Within these rows, proteins with an HMW will be found near the top, while those with an LMW will migrate nearer to the bottom of the gel.

A clear advantage of BN-PAGE compared to other methods lies in its direct analysis of endogenous proteins and its global approach. No genetic manipulation is required. This is not the case for the popular Y2H system, TAP, or the FRET/BRET technology approaches. The Y2H approach, in particular, is known to create a considerable number of FP results, probably due to analysis outside the normal environment of the interactions under investigation. The analysis of each potential PPI using these techniques also requires an independent experiment. BN-PAGE, in contrast, allows the investigation of a multitude of protein interactions by a single experiment and therefore can be considered as a large-scale approach to this question. It also allows an assessment of the MW of the complex and its abundance. Limitations of BN-PAGE compared to the above-mentioned techniques are discussed in Box 38.1.

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FIGURE 38.1. 2D BN/SDS-PAGE separation of Arabidopsis mitochondrial protein complexes. Solubilization of the complexes has been accomplished using digitonin in a ratio of 10g detergent per g protein prior to BN PAGE. Dotted lines indicate protein complexes resolved into their subunits. Arrows on top and to the left of the gel show the direction of separation in first and second dimension from high to low MW. The identity of protein complexes is given below the gel. I1-III2: supercomplex consisting of a single copy of NADH dehydrogenase (complex I) and the dimeric version of Cytochrome c reductase (complex III). I1: complex I. F1F0: mitochondrial ATP synthase (complex V). III2: dimeric complex III. F1: Soluble part of complex V. Note: Breakdown of respiratory protein complexes is observed very rarely with the use of digitonin. If it does, it might indicate that the detergent and/or Coomassie concentrations in the sample were too high.

FIGURE 38.1. 2D BN/SDS-PAGE separation of Arabidopsis mitochondrial protein complexes. Solubilization of the complexes has been accomplished using digitonin in a ratio of 10g detergent per g protein prior to BN PAGE. Dotted lines indicate protein complexes resolved into their subunits. Arrows on top and to the left of the gel show the direction of separation in first and second dimension from high to low MW. The identity of protein complexes is given below the gel. I1-III2: supercomplex consisting of a single copy of NADH dehydrogenase (complex I) and the dimeric version of Cytochrome c reductase (complex III). I1: complex I. F1F0: mitochondrial ATP synthase (complex V). III2: dimeric complex III. F1: Soluble part of complex V. Note: Breakdown of respiratory protein complexes is observed very rarely with the use of digitonin. If it does, it might indicate that the detergent and/or Coomassie concentrations in the sample were too high.

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