Protein Protocols Protein Protocols

244 Daban et al.
Fig. 1. Structure of the noncovalent hydrophobic dye Nile red.
structures the polypeptide chain is mostly situated at the interface between the
hydrocarbon core and the sulfate groups of the SDS micelles dispersed along the
unfolded protein molecule.
The enhancement of Nile red fluorescence observed with different SDS–protein
complexes occurs at SDS concentration lower than the critical micelle concentration of
this detergent in the typical Tris–glycine buffer used in SDS-PAGE (3). Thus, for Nile
red staining of SDS-polyacrylamide gels (4), electrophoresis is performed in the presence
of 0.05% SDS instead of the typical SDS concentration (0.1%) used in current
SDS-PAGE protocols. This concentration of SDS is high enough to maintain the
stability of the SDS–protein complexes in the bands, but is lower than SDS critical
micelle concentration and consequently precludes the formation of pure detergent
micelles in the gel (4,5). The staining of these modified gels with Nile red produces
very high fluorescence intensity in the SDS–protein bands and low background fluorescence
(see Fig. 2). Furthermore, under these conditions (see details in Subheading 3.),
most of the proteins separated in SDS gels show similar values of the fluorescence
intensity per unit mass.
2. Materials
All solutions should be prepared using electrophoresis-grade reagents and
deionized water and stored at room temperature (exceptions are indicated). Wear
gloves to handle all reagents and solutions and do not pipet by mouth. Collect and
dispose all waste according to good laboratory practice and waste disposal regulations.
1. Nile red. Concentrated stock (0.4 mg/mL) in dimethyl sulfoxide (DMSO). This solution is
stable for at least 3 mo when stored at room temperature in a glass bottle wrapped in
aluminum foil to prevent damage by light. Handle this solution with care, DMSO is
flammable, and, in addition, this solvent may facilitate the passage of water-insoluble
and potentially hazardous chemicals such Nile red through the skin. Nile red can be
obtained from Sigma Chemical (St. Louis, MO).
2. The acrylamide stock solution and the resolving and stacking gel buffers are prepared as
described in Chapter 11.
3. 2× Sample buffer: 4% (w/v) SDS, 20% (v/v) glycerol, 10% (v/v) 2-mercaptoethanol, 0.125 M
Tris-HCl, pH 6.8; bromophenol blue (0.05% [w/v]) can be added as tracking dye.
4. 10× Electrophoresis buffer: 0. 5% (w/v) SDS, 0.25 M Tris, 1.92 M glycine, pH 8.3 (do
not adjust the pH of this solution).