SYPRO® Ruby Protein Gel Stain (S4942) - Datasheet - Sigma

SYPRO® Ruby Protein Gel Stain (S4942) - Datasheet - Sigma
SYPRO Ruby Protein Gel Stain
Product Number S 4942
Store at Room Temperature
Product Description
SYPRO Ruby Protein Gel Stain is an organometallic
ruthenium chelate stain designed for proteomics
applications. It is a sensitive and versatile protein stain
for easy and simple detection of proteins in 1D and 2D
SDS-polyacrylamide electrophoresis gels and in IEF
gels. After staining proteins can be removed from the
gel and analyzed by post-electrophoretic procedures,
such as Edman-based sequencing and mass
spectrometry without interference from the stain.1,2
SYPRO Ruby will detect glycoproteins, calcium-binding
proteins, lipoproteins, fibrillar proteins, and other
difficultly stained proteins without staining nucleic
acids.1 As little as 1-2 ng protein/band can be detected,
similar to that of silver staining, but greater sensitivity
than Coomassie® Brilliant Blue.1-3
The staining procedure is easy and ideal for high
throughput. Gels cannot be overstained. After a quick
rinse in 7.5% acetic acid gels can be photographed.
Use a blue-light transilluminator, UV transilluminator, or
laser scanner for detection. Fluorescence intensity is
linear over three orders of magnitude of protein
quantity, greater than either silver stain or Coomassie.
Compared with silver stain the patterns obtained with
SYPRO Ruby are similar, but not identical.3
Initially intended for 2D electrophoresis applications,
SYPRO Ruby can also be used to stain Tris-glycine
SDS-polyacrylamide electrophoresis gels and IEF
gels.4 SYPRO Ruby is compatible with gels attached to
plastic backings.
SYPRO Ruby does not covalently bind to proteins. It
associates with primary amines in proteins at acidic pH.
Strongest interaction is with Lys, Arg, and His residues
and less strongly with Tyr and Trp residues.4
The detection limit of SYPRO Ruby can be as low as
1 ng/band or approximately 0.12 ng/mm2. The most
sensitive detection is obtained using a photographic
camera, CCD, or laser scanner image analysis system.
The dye has optimal excitation at 302 and 470 nm with
an emission maximum at approximately 610 nm.
A 300 nm UV or blue-light transilluminator, or laser
scanner (488 nm or 532 nm, both with a 640 nm band
pass filter) is used for detection.
SYPRO Ruby Protein Gel Stain does not interfere with
later applications for the proteins. After documenting
the stained gel may be treated with protease to prepare
the samples for matrix-assisted laser desorption
ionization-time of flight (MALDI-TOF) mass
spectrometry.2,5 Proteins from stained gels can also be
analyzed by Edman microsequencing procedures.5
Reagent
SYPRO Ruby Protein Gel Stain is provided as a readyto-use solution. For optimal sensitivity further dilution is
not necessary or recommended.
Storage/Stability
Store at room temperature protected from light. The
solution is stable at least nine months.
Procedure
Gel Staining
1. Fixing the gel:
a. 2D gels: fix gels for 30 minutes in a solution of
methanol or ethanol and acetic or
trichloroacetic acid. Examples are 10%
methanol with 7% acetic acid; 25% ethanol with
12.5% trichloroacetic acid; or 50% ethanol with
3% acetic acid.
b. IEF gels: fix for three hours in a solution of 40%
methanol with 10% trichloroacetic acid. Wash
three time in water for 10 minutes each.
c. 1D gels: Fixing not required before staining.
2. Pour stain into a clean, detergent-free
polypropylene or polyvinyl chloride staining dish.
Do not use glass dishes. Use approximately 50 ml
for 8 × 10 cm (0.57 mm thick) gels to 500 ml for
20 × 20 cm (1 mm thick) gels. For larger gels use a
volume of staining solution equal to approximately
10 times the gel volume. Using too little staining
solution can reduce sensitivity.
3. Place gel in staining solution and cover with
aluminum foil to protect from light during staining.
4. Place on a platform shaker for gentle agitation for
at least three hours for 1D and 2D gels. IEF gels
should be stained overnight. Gels will not overstain
if left in staining solution overnight. Reuse of
staining solution is not recommended because
significant loss of sensitivity will result.
5. Washing the gels:
a. Transfer gel to a clean staining dish. Wash gels
in 10% methanol or ethanol containing 7%
acetic acid for 30 minutes. IEF gels should be
washed three times. Transferring to a new dish
reduces fluorescent specks on the gel and
washing improves sensitivity by reducing
background fluorescence.
b. Alternatively gels may be washed in water, but
background fluorescence is not reduced as
much.
6. Place gel directly on the transilluminator for
photographing. Do not use plastic wrap because it
will autofluoresce more than normal in the presence
of SYPRO Ruby. If the gel has a plastic backing it
should be removed if it autofluoresces. It may also
bind the dye resulting in high background.
Detection:
SYPRO Ruby has excitation maxima at approximately
280 nm and 450 nm. For detection either a 300-nm UV
transilluminator or a blue-light transilluminator can be
used. Laser imaging systems with emissions at 450,
473, 488, or 532 nm can also be used.
Photography:
A photographic or CCD camera with appropriate filters
attached will provide the greatest sensitivity.
For photography use Polaroid Type 667 (Product No.
F 4638) or Type 57 (Product No. F 4513) film and the
SYPRO Photographic Filter (Product No. S 6067). As a
starting exposure use an f-stop of 4.5 and an exposure
time of 1 second. Adjust these settings as needed to
obtain optimal results. Transilluminators may have
different light intensities depending on brand of
instrument and age of bulbs. Other film types have
lower film speeds requiring much longer exposures and
possibly a different filter. Long exposures under UV
illumination can be made, if needed, because SYPRO
Ruby is very photostable.
References
1. Berggren, K., et al., Background-free, high
sensitivity staining of proteins in one- and twodimensional sodium dodecyl sulfate-polyacrylamide
gels using a luminescent ruthenium complex.
Electrophoresis, 21, 2509-2521 (2000).
2. Lopez, M. F., et al., A comparison of silver stain
and SYPRO Ruby Protein Gel Stain with respect to
protein detection in two-dimensional gels and
identification by peptide mass profiling.
Electrophoresis, 21, 3673-3683 (2000).
3. Görg, A., et al., The current state of twodimensional electrophoresis with immobilized pH
gradients. Electrophoresis, 21, 1037-1053 (2000).
4. Steinberg, T. H., et al., Ultrasensitive fluroescence
protein detection in isoelectric focusing gels using a
ruthenium metal chelate stain. Electrophoresis, 21,
486-496 (2000).
5. Berggren, K., et al., A luminescent ruthenium
complex for ultrasensitive detection of proteins
immobilized on membrane supports. Anal.
Biochem., 276, 129-143 (1999).
SYPRO is a registered trademark of Molecular Probes,
Inc., Eugene, OR.
Coomassie is a registered trademark of Imperial
Chemicals Industries PLC.
GL/MAM/JWM 06/04
Sigma brand products are sold through Sigma-Aldrich, Inc.
Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser
must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side of
the invoice or packing slip.
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