Symbiotic Fungi: Principles and Practice (Soil Biology)

258 E. Dumas-Gaudot et al.
solubilized in electrophoresis buffer and previously stained with a little bromophenol
blue.
5. Place each gel cassette into the tank (Hoefer DALT II system, AB) and fill with
the electrophoresis SDS buffer up to cover the whole gel surface.
6. Run electrophoresis at 10 C for 30 min at 30 V, then 30 min at 60 V and finally
at 100 V overnight until the dye front reaches the bottom of the gels.
15.4.6 Protein Detection
The application of the 2-DE technology to separate, analyze and characterize
proteins of biological samples would not have been possible without the development
of complementary detection methods. The following non-radioactive stains
can be employed for protein detection: metal stain (zinc/copper), Coomassie blue,
silver and fluorescent dies. Perhaps one of the most popular is the silver-staining
method, which is 100-fold more sensitive than Coomassie Brilliant Blue staining
(Chevalier et al. 2004; Lopez et al. 2000; Rabilloud 2001). However, there can be
some problems using this stain as a quantitative procedure, because it is known to
be non-stoechiometric and prone to saturation and negative staining effects, where
regions of very high protein concentration do not stain and appear as ‘holes’ in the
pattern of stained spots. Moreover, only a few silver-staining methods are compatible
with further MS analyses (Rabilloud 2001). Finally, detection methods based on
fluorescent compounds promise to overcome these problems because of their
excellent linearity and high dynamic range (i.e., they can be used over a wide
range of protein concentrations) (Chevalier et al. 2004).
15.4.6.1 Colloidal Coomassie Blue Staining
This stain is not specific, and will also detect non-protein components such as
polysaccharides. Its sensitivity is fairly good, and it is compatible with mass
spectroscopy. We use the protocol of Mathesius et al. (2001), and find it easy to
use and quite sensitive enough. It can detect 36–47 ng of protein.
1. After electrophoresis rinse gels briefly (