Bio-Rad Total Protein Blot Stains - Manual (Page 6)

PART 3: SYPRO Ruby protein blot stain
Section 1
Introduction
1.1 General Information
SYPRO Ruby protein blot stain is a ready-to-use, sensitive,
f l u o r e s cent stain for detecting proteins on nitrocellulose or
polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby protein
blot stain is a permanent protein stain that can be used to stain proteins
on membrane supports prior to antibody- or lectin- mediated
visualization of specific proteins. The stain can also be used prior to
protein identification by Edman-based sequencing or Mass
Spectrometry. Since SYPRO Ruby protein blot stain does not
b l o c k e p i topes, it can be used in conjunction with standard
immunological procedures, eliminating the need for duplicate blots.
This compatibility is particularly advantageous in blots of 2-D gels,
where precise localization of the targeted protein relative to
other proteins is often difficult.
1.2 Product Description
SYPRO Ruby protein blot stain is a 1x pre-mixed solution, provided
with instructions. The 200 ml volume is sufficient for staining 10–40
mini gel electroblots, or ~ 4 large-format electroblots.
170-3127 SYPRO Ruby protein blot stain, 200 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 forceps to handle wet blots,
since residue found on latex gloves may destroy the staining pattern.
Once dry, membranes can be handled freely.
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a) UV or blue-light transilluminator
Gels that contain proteins stained with SYPRO Ruby protein gel
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 cam-
era system or CCD-based systems, a yellow filter such as the
yellow Wratten 9 must be utilized for image collection. Typical
Polaroid camera settings for capturing an image of a minigel
stained with SYPRO Ruby protein gel stain are f/4.5 or better at a
1-second exposure setting.
• The Bio-Rad Gel Doc™ 1000, 2000, and ChemiDoc™ CCD-
based camera systems are equipped with a UV transilluminator
for imaging SYPRO Ruby protein gel stain.
• Bio-Rad’s Fluor-S™ and Fluor-S MAX MultiImagers are
equipped with trans-scanning UV for visualizing and imaging
SYPRO Ruby stained gels. Use the Transmission Mode and
550LP filter.
• For other Polaroid-based or CCD-based systems please contact
your camera manufacturer for appropriate recommendations on
use of filter sets with SYPRO Ruby protein gel stain.
b) Laser-based instruments
• SYPRO Ruby protein gel stain can be imaged using laser-based
imaging systems equipped with 450, 473, 488 or 532 nm laser
lines. The following laser/filter combinations are recommended:
488/550LP; 488/605DF50; 532/550LP; 532/605DF50.
The Bio-Rad Molecular Imager™ FX also has a pre-pro-
grammed function; select Protein Stain Gel/SYPRO Ruby from
the Application Menu to automatically select the correct laser
and filter sets for this stain.
• For other laser-based imaging systems please contact the
manufacturer of the instrument for recommended use of filter
sets with SYPRO Ruby protein stain.
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