Bio-Rad Total Protein Blot Stains - Manual (Page 8)

b) Wash the gel in deionized water 4 times for 30 min each to
decrease background fluorescence. The gel may be monitored
periodically using UV epi-illumination to determine the level of
background fluorescence.
Note: IEF gels with plastic backings separate from their backing
during this water wash. It is important to remove this backing as it
has high inherent fluorescence.
2.2 Viewing and Imaging an IEF Gel
SYPRO Ruby protein stain is excited around its two excitation
peaks at ~ 280 nm and ~ 450 nm and has an emission maxima at ~ 610
nm. Stained proteins can be visualized using a variety of excitation
sources including a 300 nm UV or blue-light transilluminator, or laser-
based systems. SYPRO Ruby protein stain also has exceptional photo-
stability and a long emission lifetime, allowing for long exposure times
while minimizing background fluorescence.
a) UV or blue-light transilluminator
Gels that contain proteins stained with SYPRO Ruby IEF stain are
readily visualized using a UV or blue light source. The use of a
photographic camera or CCD camera and the appropriate filters is
essential to obtain the greatest sensitivity with these systems.
• It is important to clean the surface of the transilluminator before
each use with deionized water and a soft, lint free cloth to
remove stains from the glass (including SYPRO, SYBR and
EtBr stain) that can lead to high background or skewed data.
• When capturing an image using a standard Polaroid camera
system or CCD-based systems, a yellow filter such as the yellow
Wratten 9 must be used. Typical Polaroid camera settings for
capturing an image of a minigel stained with SYPRO Ruby IEF
stain are f/4.5 or better at a 1-second exposure setting.
• Bio-Rad’s Gel Doc™ 1000, 2000, and ChemiDoc™ CCD-based
camera systems are equipped with a UV transilluminator for
imaging SYPRO Ruby IEF stained gels.
• Bio-Rad’s Fluor-S™ and Fluor-S MAX MultiImagers are
equipped with trans-scanning UV for visualizing and imaging
SYPRO Ruby IEF stain. Use the Transmission Mode and 550LP
filter.
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Part 4: SYPRO Ruby IEF stain
Section 1
Introduction
1.1 General Information
SYPRO Ruby IEF stain is a ready–to–use, sensitive, fluorescent
stain for detecting proteins separated in isoelectric focusing (IEF) gels.
The stain does not interfere with subsequent analysis of proteins by
Edman-based sequencing or mass spectrometry and is quantitative over
three orders of magnitude. Use SYPRO Ruby IEF stain for difficult to
stain proteins such as glycoproteins and lipoproteins. This stain will not
stain nucleic acids.
1.2 Product Description
SYPRO Ruby IEF stain is a 1x pre-mixed solution, supplied with
instructions. The 400 ml size will stain ~10 IEF mini gels or 2 standard
flat-bed IEF gels.
170-3148 SYPRO Ruby IEF stain, 400 ml
Section 2
Instructions
2.1 Staining
Perform all washing and staining steps at room temperature in a
clean plastic container (see Part 1, Section 1.4), with continuous, gentle
agitation (e.g. on an orbital shaker). Use plastic, rather than latex, gloves
to reduce the amount of smudging on the gel.
Note: RUBY IEF Stain contains a fixative; no fixation is required.
a) Incubate the gel in SYPRO Ruby IEF stain overnight (12–18
hrs)
Gel Size Volume of Stain
6 x 9 cm 40 ml
8 x 10 cm 50 ml
24.5 x 11 cm 200 ml
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SYPRO Ruby IEF stain SYPRO Ruby IEF stain
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