Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 53)

Table 11.1. Flamingo fluorescent gel stain.
Volume of fixing Volume of staining
Gel size solution per gel solution per gel
Mini (8.6 × 6.8 cm) 100 ml 50 ml
Midi (13.3 × 8.7 cm) 200 ml 100 ml
Large 500 ml 250 ml
(16 × 16 cm or 16 × 20 cm)
Larger (25.6 × 23 cm)
1,000 ml 500 ml
Fig. 11.1. 2-D gel stained with Bio-Safe Coomassie stain.
Fig. 11.2. 2-D gel stained with Flamingo stain.
102 103
2-D Electrophoresis Guide Methods Chapter 11: Protein Detection
Wash gels three times for 5 min each
in distilled or deionized H
2
O.
Remove water from staining container
and add Bio-Safe Coomassie stain to
completely cover the gel. Agitate for
at least 1 hr.
Rinse in distilled or deionized H
2
O for
at least 30 min. Stained gels can be
stored in water.
Place gel in a staining tray with fixing
solution (40% ethanol, 10% acetic acid).
Cover the tray and agitate gently for
at least 2 hr.
Pour off the fixing solution and add
1× stain solution (dilute 1 part Flamingo
fluorescent gel stain with 9 parts
deionized or distilled H
2
O). Cover the
tray and agitate gently. Stain for at
least 3 hr.
Optional background reduction:
Carefully pour off the stain solution
and replace with an equal volume of
0.1% (w/v) Tween 20. Cover the tray and
agitate gently for 10 min.
Rinse gel with deionized or distilled
H
2
O prior to imaging.
Bio-Safe Coomassie Stain
Instruction manual: bulletin 4307051.
Protocol
For more detailed instructions, refer to the
respective instruction manuals. Flamingo Fluorescent Gel Stain
Instruction manual: bulletin 10003321. Refer to
Table 11.1 for solution volumes.
Protocol
Total Protein Staining
1
1
2 2
3
3
4
Long-Term Storage of Stained Gels
Gels stained with a visible stain can serve as a
permanent record of the SDS-PAGE separation.
Stained gels may be stored indefinitely when dried
between cellophane sheets. To dry stained gels,
the gel is placed on a sheet of wet cellophane.
A second sheet of wet cellophane is carefully laid
over the gel with care taken not to introduce bubbles
or wrinkles. The gel, sandwiched between two
sheets of wet cellophane, is clamped into a frame
and allowed to dry.
The most common problem associated with drying
gels is cracking. Cracking is best prevented by
soaking the gel for at least 30 min in a 2% (w/v)
solution of glycerol in water prior to drying.
Alternatively, a commercially available gel-drying
solution may be used.
■ ■
Fluorescent dyes like Flamingo and Oriole ™
fluorescent gel stains have a higher dynamic range
than Coomassie (Brilliant) Blue or silver staining
techniques and are, therefore, recommended for
quantitative protein analysis
■ ■
Gels stained with fluorescent dyes can be
counterstained with Bio-Safe
™ Coomassie stain
for further reference and to enhance sensitivity of
the Coomassie stain
■ ■
Silver staining is not generally recommended
when protein spots will be identified by mass
spectrometry, though some formulations are
compatible with mass spectrometry at the
expense of promised sensitivity. Use Bio-Safe
Coomassie or fluorescent dyes like Flamingo
or Oriole instead
■ ■
As an alternative to drying gels, seal them in
zip-top plastic bags in either water or, for long-term
storage, water with 0.005% sodium azide. Fill the
bag with water, insert the gel, expel the water,
and seal the bag
Tips for Total Protein Staining
■ ■
Stain gels at room temperature with gentle
agitation (for example, on an orbital shaker),
making sure the gel is completely covered
with stain solution at all times
■ ■
Use any convenient glass or plastic container
that is appropriate to the method chosen. Use
glass containers with silver staining methods
or with Flamingo
™ stain. Use plastic trays with
SYPRO Ruby stain
■ ■
Use Bio-Rad’s Dodeca ™ stainers for
high-throughput staining
■ ■
Wear gloves during the staining process, and
handle gels only by the edges and corners.
Wet gloves with water or buffer before handling
the gel to keep the gel from sticking and tearing
■ ■
Use clean and dust-free containers for gel
staining. Place a lid on the container to avoid
contamination of the staining solution
■ ■
Use pure chemicals and highly purified water
(conductivity <2 μS)
■ ■
When performing gel staining with
fluorescent dyes, cover the staining tray
with foil during incubations