Bio-Rad Total Protein Blot Stains - Manual (Page 7)

2.2 Visualizing and Imaging a SYPRO Ruby stained
blot
RUBY BLOT stain has two excitation peaks at ~ 280 nm and
~ 450 nm and has an emission maxima near 618 nm.
a) Epi UV Illumination
• When viewing blots stained with SYPRO Ruby protein blot
stain, only one side of the membrane can be excited due to the
non-transparency of the membrane material. Therefore, only
epi-UV illumination can effectively be used to visualize or
image the blots. Epi illuminating CCD-based imaging systems
or a hand-held UV source centered at 300 nm can be used for
excitation: When capturing images with an epi-UV illuminated
CCD-based system, a yellow filter such as the yellow Wratten
# 9 must be utilized for image collection.
• Bio-Rad’s Fluor-S™ and Fluor-S MAX MultiImagers are
equipped with epi-UV for visualizing and imaging SYPRO
Ruby protein blot stain. Use Epi-UV illumination and 610LP
emission filter.
• For other epi-UV illuminated CCD-based systems please contact
your camera manufacturer for appropriate recommendations on
use of filter sets with SYPRO Ruby protein stains.
b) Laser-based Instruments
• SYPRO Ruby protein blot stain can be imaged using laser-based
imaging systems equipped with 450, 473, 488 or 532 nm laser
lines that use epi illumination and collection. For imaging blots
with SYPRO Ruby protein blot stain on the Bio-Rad Molecular
Imager™ FX use either the 488 or the 532 laser in combination
with the 640DF35 filter. For other epi illuminated laser-based
imaging systems please contact the manufacturer of the
instrument for recommended use of filter sets with SYPRO
Ruby protein stains.
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a) Staining Nitrocellulose Membranes
1) After electroblotting proteins to a nitrocellulose membrane,
completely immerse the membrane in 7% acetic acid, 10%
methanol and incubate for 15 minutes.
2) Wash the membrane in deionized water 4 times for 5 minutes
each.
3) Completely immerse the membrane in SYPRO Ruby protein
blot stain for 15 minutes.
4) Wash the membrane in deionized water 4 to 6 times for
1 minute each to remove excess stain. The membrane may be
monitored periodically using UV epi-illumination to
determine the level of background fluorescence.
5) Membranes stained with SYPRO Ruby protein blot stain are
best preserved by allowing membranes to air dry.
b) Staining PVDF Membranes
1) After electroblotting proteins to a PVDF membrane, allow
the membrane to dry completely.
2) Float the membrane face down in 7% acetic acid,
10% methanol and incubate for 15 minutes.
3) Wash the membrane in deionized water 4 times for 5 minutes
each.
4) Transfer the blot using forceps to a staining dish containing
SYPRO Ruby protein blot stain.
5) Float the membrane in SYPRO Ruby protein blot stain for 15
minutes.
6) Wash the membrane in deionized water 2 to 3 times for
1 minute each to remove excess stain. The membrane may be
monitored periodically using UV epi-illumination to
determine the level of background fluorescence.
7) Membranes stained with SYPRO Ruby protein blot stain are
best preserved by allowing membranes to air dry. After air
drying they can be handled without using forceps.
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SYPRO Ruby protein blot stain SYPRO Ruby protein blot stain
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