40
SYPRO
®
Ruby Staining Protocol
Introduction
Instructions for staining E-PAGE
™
Gels using SYPRO
®
Ruby Protein Gel Stain is
described in this section. To visualize protein bands after electrophoresis using
SYPRO
®
Ruby, you will need a UV transilluminator or a laser-based scanner (see
below). For further details on imaging SYPRO
®
Ruby proteins, refer to the
product manual available at
Support (page 72).
Total staining time is 4 hours, with optional destaining
overnight.
Materials Needed
You will need the following items for staining one E-PAGE
Gel. See page 71
for ordering information.
•
SYPRO
®
Ruby Protein Gel Stain (see note below)
•
Fixing solution (20% acetic acid, see note below)
•
Destaining solution (10% methanol, 7% acetic acid, see note below)
•
Incubation Trays
•
Rotary shaker
•
UV transilluminator equipped with a standard camera or an appropriate
laser scanner (see note below)
The volume of fixing, staining and destaining solutions will depend on the
volume of your staining container. To obtain good results, the volume of solution
must be sufficient to cover the gel completely and to allow the gel to move freely
during all of the steps.
Staining Protocol
1.
After electrophoresis, remove the gel from the cassette (page 24) and place
the gel in a clean Incubation Tray.
2.
Fix the gel in 20% acetic acid for 30 minutes on an orbital shaker.
3.
Stain the gel in undiluted SYPRO
®
Ruby Protein Gel Stain for 1.5 hours on an
orbital shaker.
4.
Transfer the gel to a clean Incubation Tray and destain in 10% methanol, 7%
acetic acid for 2 hours. If complete removal of background is desired,
perform the destaining step overnight.
5.
Place the gel on a UV transilluminator equipped with a standard camera and
select the ethidium bromide filter on the camera.
You may also use a laser-based scanner with a laser line that falls within the
excitation maxima of the stain (610 nm).
6.
Image the gel with a suitable camera with the appropriate filters using a
1–4 second exposure. You may need to adjust the brightness and contrast to
reduce any faint non-specific bands.
You should see fluorescent protein bands and the gel should have minimal
background as shown on page 55.
Summary of Contents for E-PAGE Gels
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