Comparative Analyses of Protein Complexes by Blue Native DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 146

fl uorophore detection allows exact relative quantifi cation of
proteins present in the protein fractions to be compared.
Classically, DIGE is carried out based on the two-dimensional
(2D) isoelectric focusing/sodium dodecyl sulfate polyacrylamide
gel electrophoresis (IEF/SDS PAGE) system ( 1 ). Indeed, using
this experimental system, large protein sets are effi ciently resolved.
However, 2D IEF/SDS PAGE also has limitations. For instance,
hydrophobic proteins are not well resolved during the IEF gel
dimension, and proteins with very basic isoelectric points (pIs)
often get lost. Additionally, 2D IEF/SDS PAGE is not compatible
with native enzyme characterizations. 2D blue native (BN)/SDS
PAGE represents an alternative gel system which allows overcoming
these limitations. Hydrophobic as well as basic proteins are easily
resolved. If applied as a one-dimensional system, blue native poly-
acrylamide gel electrophoresis (BN PAGE) is even compatible with
in-gel enzyme activity stainings.
BN PAGE was fi rst published by Hermann Schägger ( 2 ). The
principle idea was to use Coomassie Blue not only for protein stain-
ing after gel electrophoresis but also for the introduction of nega-
tive charges into proteins by incubating protein fractions with this
compound before gel electrophoresis. Coomassie Blue is an anionic
molecule but, in contrast to SDS, does not denature proteins.
Furthermore, protein complexes remain stable. If combined with
low-percentage polyacrylamide gels, protein complexes can be
resolved by BN PAGE. If sample preparation takes place in the
presence of mild nonionic detergents, even hydrophobic mem-
brane-bound protein complexes can easily be separated. As with
IEF/SDS PAGE, strips of blue native gels can be horizontally
transferred onto a second gel dimension which is carried out in the
presence of SDS. Using this experimental setup, protein complexes
are separated into their subunits which form vertical rows of spots
on the resulting 2D gels.
BN PAGE and 2D blue native/sodium dodecyl sulfate poly-
acrylamide gel electrophoresis (BN/SDS PAGE) can be combined
with the DIGE technology ( 3 ). BN DIGE allows systematic
and quantitative comparison of protein complexes of related
protein fractions, structural investigation of protein complexes,
assignment of protein complexes to subcellular fractions like
organelles, electrophoretic mapping of isoforms of subunits of
protein complexes with respect to a larger proteome, and topo-
logical investigations ( 4 ). So far, BN DIGE has only been applied
in a few studies ( 4-9 ). Indeed, the potential of BN DIGE was
only very recently recognized. Here we present a BN DIGE pro-
tocol suitable for the comparative analyses of protein complexes
of protein fractions.