Protein Protocols Protein Protocols

Rapid Staining with Nile Red 245
Fig. 2. Example of Nile red staining of different proteins and peptides in 0.05% SDS–15%
polyacrylamide gels. The protein molecular weight markers (lane 6, from top to bottom: BSA,
ovalbumin, glyceraldehyde-3-phosphate dehydrogenase, trypsinogen, and lysozyme), and BSA
digested with increasing amounts of trypsin (lanes 1–5), were stained for 5 min with a solution
of Nile red in water prepared by quick dilution of a stock solution of this dye in DMSO (see
Subheading 3.).
5. Plastic boxes for gel staining. Use opaque polypropylene containers (e.g., 21 × 20 × 7.5
and 12 × 7.5 × 7 cm for large [20 × 16 × 0.15 cm] and small [8 × 6 × 0.075 cm] gels,
respectively) with a close-fitting lid to allow intense agitation without spilling the staining
solution.
6. Orbital shaker for the agitation of the plastic boxes during gel staining.
7. Transilluminator equipped with midrange ultraviolet (UV) bulbs (~300 nm) to excite
Nile red (3,4). A transilluminator with a cooling fan (e.g., Foto UV 300 [Fotodyne Inc.,
Harland, WI] or similar) is very convenient to prevent thermal damage of the gel when
long exposures are necessary (see Subheading 3., step 11). UV light is dangerous to skin
and particularly to eyes. UV-blocking goggles and a full face shield, and protective
gloves and clothing, should be worn when the stained gel is examined using the transilluminator.
For the photography, we place the transilluminator with the gel and the photographic
camera inside a home-made cabinet with opaque (UV-blocking) curtains to
prevent operator exposure to UV light. Alternatively, the gel can be transilluminated
inside a compact darkroom of a documentation system provided with a charge-coupled
device (CCD) camera (e.g., Gel Doc 1000 from Bio-Rad Laboratories [Hercules, CA]).
To avoid the problems associated with UV light, we have constructed a transilluminator
that works in the visible region (~540 nm; green-light) and can also be used for the excitation
of Nile red stained bands (11).
8. Photographic camera (e.g., Polaroid [Cambridge, MA] MP-4 camera) or a CCD camera.
9. Optical filters. With the Polaroid camera and UV transilluminator use the Wratten (Kodak
[Rochester, NY]) filters number 9 (yellow) and 16 (orange) to eliminate the UV and visible
light from the transilluminator. Place the two filters together in the filter holder of the
camera so that filter 16 is on top of filter 9 (i.e., filter 16 should be facing towards the