Protein Protocols Protein Protocols

Transition Metal Chelate Stains 281
3.6. Luminescent Detection of Proteins in Gels
Using SYPRO Ruby IEF Protein Gel Stain
SYPRO Ruby IEF protein gel stain is a ready-to-use, ultrasensitive, luminescent
protein stain created especially for the analysis of proteins in IEF gels. This fluorescent
stain attains comparable sensitivity to that of the best silver-staining techniques. Staining
protocols are simple, the stain is ready-to-use, and it cannot overstain. It will not
stain extraneous nucleic acids and the stain detects glycoproteins and other difficult-tostain
proteins. It does not interfere with subsequent analysis of proteins by Edmanbased
sequencing or mass spectrometry. SYPRO Ruby IEF protein gel stain is also
compatible with agrose gels or polyacrylamide gels adhering to plastic backings.
1. Prepare and run IEF gels according to standard protocols. The staining technique is appropriate
for carrier ampholyte isoelectric focusing or immobilized pH gradient (IPG) gels (24).
Perform SYPRO Ruby IEF gel staining with continuous, gentle agitation (e.g., on an orbital
shaker at 50 rpm). For small gels, use circular staining dishes on orbital shakers if possible.
2. Clean and thoroughly rinse the staining dishes before use. (see Note 20).
3. Incubate the gel in the undiluted stain overnight. (see Note 19). Caution: SYPRO Ruby
IEF protein gel stain contains a caustic acid. The stain should be handled with care,
using protective clothing, eye protection, and gloves.
4. Rinse the gel in four changes of deionized water over 2 h to decrease background fluorescence.
The gel may be monitored periodically using UV transillumination (see Note 21)
to determine the level of background fluorescence. For IEF gels with plastic backings, the
gel separates from the backing during this water wash. It is important that the backing be
removed from the gels as it has high inherent fluorescence.
5. To dry the stained gel for permanent storage, incubate the gel in a solution of 2% glycerol
for 30 min. Dry the stained gel using a gel dryer by standard methods. Note that proteins
present at very low levels may no longer be detectable after gel drying.
6. Stained proteins in wet or dried gels may be visualized by reflective or transmissive UV
illumination or using a laser-based gel scanner (see Note 21).
3.7. Elution of Metal Chelate Stains
The Ferrozine-ferrous, Pyrogallol Red–molybdate, Ferrocyanide–ferric, colorimetric
metal chelate stains as well as the luminescent bathophenanthroline disulfonate–
europium and SYPRO Rose Plus protein blot stains are readily removed from
electroblotted proteins by incubation in mildly alkaline solution. SYPRO Ruby protein
blot stain is not readily reversible by similar methods, although it is slowly removed
from proteins during blocking and incubation steps commonly used in immunoblotting
procedures. The gel stains are also not readily destained.
The Ferrozine–ferrous stain can be eluted by immersing the blots in 50 mM Tris-HCl,
pH 8.8, 200 mM NaCl, 20 mM EDTA for 15 min on a rotary shaker (50 rpm). Blots
treated with the Ferrozine–ferrous stain followed by enhancement with the ferrocyanide–
ferric stain require harsher elution conditions. This stain is eluted by incubation in 200 mM
sodium carbonate, 100 mM EDTA, pH 9.6 for 10 min. As the bathophenanthrolineeuropium
and SYPRO Rose Plus stains can be observed only upon UV illumination,
they do not require elution if subsequent colorimetric detection procedures are to be
performed (29). If concerned about interference with subsequent procedures, however,
the stains can be eluted from blots by incubation in 200 mM sodium carbonate, 100 mM