Protein Protocols Protein Protocols

Quantification of Proteins on Gels 239
2.2. Sypro Ruby Method
1. Equipment for scanning fluorescent bands on gels, for example, the Bio-Rad Fluor S
MultiImager with Multi Analyst software, which can be used to photographically record
or scan wet gels, allowing repeated scanning procedures (as staining or destaining proceeds).
Simple viewing of stained bands may be done under a hand-held 300-nm UV lamp
or on a transilluminator. Protect eyes with UV-opaque glasses. The Sypro dye may be
excited at 280 or 450 nm and emits at 610 nm.
2. Protein stain: Sypro Ruby gel stain (Molecular Probes, product number S-12000 or S-12001)
(see Note 2). This stain is stable at room temperature in the dark. Exact details of the stain
are not revealed but according to the manufacturer, the stain contains neither hazardous nor
flammable materials. Use fresh, undiluted stain only.
3. Destain: Background may be reduced by rinsing in water.
4. Clean dishes, free of dust that might contribute to background staining.
3. Method
3.1. Colloidal Coomassie Brilliant Blue G Method
1. At the end of electrophoresis, wash the gel for a few minutes with several changes of
water (see Note 3), then immerse the gel (with gentle shaking) in the colloidal Coomassie
Brilliant Blue G. This time varies with the gel type (e.g., 1.0–1.5 h for a 1–1.5 mm thick
sodium dodecyl sulfate [SDS] polyacrylamide gel slab), but cannot really be overdone.
Discard the stain after use, for its efficacy declines with use.
2. At the end of the staining period, decolorize the background by immersion in distilled
water, with agitation, and a change of water whenever it becomes colored. Background
destaining is fairly rapid, giving a clear background after a few hours (see Notes 4 and 5).
3. Measure the extent of blue dye bound by each band by scanning densitometry. Compare
the dye bound by a sample with those for standard proteins run and stained in parallel with
the sample, on the same gel (see Notes 6–8 and 11–17).
3.2. Sypro Ruby Method
1. At the end of electrophoresis, rinse the gel in water briefly, put it into a clean dish and then
cover it with Sypro Ruby gel stain solution. Gently agitate until staining is completed, which
may take up to 24 h or longer (see Note 9). Overstaining will not occur during prolonged
stained. Do not let the stain dry up on the gel during long staining procedures. Discard the
stain after use, for it becomes less efficacious with use. During the staining procedure the gel
may be removed from the stain and inspected under UV light to monitor progress. If the
staining is insufficient, the gel may be replaced in the stain for further incubation.
2. Destain the background by washing the gel in a few changes of water for 15–30 min.
3. Measure the extent of dye bound, that is, the luminescence, by each band by scanning.
Compare the dye bound by a sample with that for standard proteins run and stained in
parallel with the sample, on the same gel (see Note 10).
4. Notes
1. The Coomassie Brilliant Blue G stain mixture may be readily made from the components.
A commercially available alternative is the Gel Code blue stain reagent from Pierce (product
no. 24590 or 24592). Details of the stain components are not divulged, other than they
also include Coomassie (G250), but the stain is used in the same way as described for the
Sigma reagent, gives similar results, and costs approximately the same.