Protein Protocols Protein Protocols

Rapid Staining with Nile Red 243
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Rapid and Sensitive Staining of Unfixed Proteins
in Polyacrylamide Gels with Nile Red
Joan-Ramon Daban, Salvador Bartolomé
Antonio Bermúdez, and F. Javier Alba
1. Introduction
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is one of
the most powerful methods for protein analysis (1,2). However, the typical procedures
for the detection of protein bands after SDS-PAGE, using the visible dye
Coomassie Blue and silver staining, have several time-consuming steps and require the
fixation of proteins in the gel. This chapter describes a rapid and very simple method
for protein staining in SDS gels developed in our laboratory (3–5). The method is based
on the fluorescent properties of the hydrophobic dye Nile red (9-diethylamino-5Hbenzo[α]phenoxazine-5-one;
see Fig. 1), and allows the detection of < 10 ng of unfixed
protein per band about 5 min after the electrophoretic separation. Furthermore, it has
been shown elsewhere (6,7) that, in contrast to the current staining methods, Nile red
staining does not preclude the direct electroblotting of protein bands and does not
interfere with further staining, immunodetection and sequencing (see Chapter 50).
Nile red was considered a fluorescent lipid probe, because this dye shows a high
fluorescence and intense blue shifts in presence of neutral lipids and lipoproteins (8).
We have shown that Nile red can also interact with SDS micelles and proteins
complexed with SDS (3). Nile red is nearly insoluble in water, but is soluble and shows
a high increase in the fluorescence intensity in nonpolar solvents and in presence of
pure SDS micelles and SDS–protein complexes. In the absence of SDS, Nile red can
interact with some proteins in solution but the observed fluorescence is extremely
dependent on the hydrophobic characteristics of the proteins investigated (8,9). In
contrast, Nile red has similar fluorescence properties in solutions containing different
kinds of proteins associated with SDS, suggesting that this detergent induces the formation
of structures having equivalent hydrophobic properties independent of the different
initial structures of native proteins (3). In agreement with this, X-ray scattering
and cryoelectron microscopy results have shown that proteins having different properties
adopt a uniform necklace-like structure when complexed with SDS (10). In these
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
243