Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 54)

Table 11.3. SYPRO Ruby fluorescent gel stain.
Gel size
Volume of staining solution per gel
8 × 108 cm 50 ml
16 × 20 cm 330 ml
20 × 20 cm 500 ml
Table 11.4. Silver Stain Plus.
Gel Thickness
0.75–1.0 mm
Step Time Mini Gel Large Gel
Fixative* 20 min 400 ml 800 ml
Water washes 10 min 400 ml 800 ml
Stain** 20 min 100 ml 300 ml
Stop 15 min 400 ml 400 ml
Gel Thickness
1.5–3.0 mm
Step Time Mini Gel Large Gel
Fixative* 30 min 400 ml 800 ml
Water washes 20 min 400 ml 800 ml
Stain** 20 min 100 ml 300 ml
Stop 15 min 400 ml 400 ml
* Gels may be left in this solution indefinitely prior to staining;
therefore, it is not necessary to carry out the entire procedure
directly following electrophoresis.
** Stain until the desired intensity is reached. It may take at least
15 min before the first bands or spots become visible. Staining time
is dependent on the sample and quantity loaded.
Table 11.2. Oriole fluorescent gel stain.
Gel size
Volume of staining solution per gel
Mini (8.6 × 6.8 cm) 50 ml
Midi (13.3 × 8.7 cm) 100 ml
Large (16 × 16 cm or 16 × 20 cm) 250 ml
Larger (25.6 × 23 cm) 500 ml
Fig. 11.5. 2-D gel stained with Silver stain.
Fig. 11.3. 2-D gel stained with Oriole stain.
Fig. 11.4. 2-D gel stained with SYPRO Ruby stain.
104 105
2-D Electrophoresis Guide Methods Chapter 11: Protein Detection
Wash the gel in one of the following
gel fixing solutions for 30 min:
■ ■
10% methanol, 7% acetic acid
■ ■
25% ethanol, 12.5% trichloroacetic acid
■ ■
10% ethanol, 7% acetic acid
■ ■
50% ethanol, 3% acetic acid
■ ■
40% ethanol, 10% acetic acid
Remove the wash solution and cover
the gel with SYPRO Ruby protein gel
stain. In general, use ~10 times the
volume of the gel. Using too little
stain will reduce sensitivity.
Stain the gel with continuous gentle
agitation for at least 3 hr for best
sensitivity. Specific staining can be
seen in 30–90 min. For convenience,
gels can be left in the stain solution
overnight (16–18 hr).
Rinse the gel in 10% methanol
(or ethanol), 7% acetic acid for
30–60 min to decrease background
fluorescence. Rinse the gel in water
before imaging.
Prepare the development accelerator
reagent solution. Add the entire
contents (50 g) of development
accelerator reagent to deionized
distilled H
2
O and bring volume up to 1 L.
Store at 4°C and use within 3 months.
Fixative step. Make fixative enhancer
solution by mixing 50% (v/v) reagent-
grade methanol, 10% (v/v) reagent-grade
acetic acid, 10% (v/v) fixative enhancer
concentrate, and 30% (v/v) deionized
distilled H
2
O. After gel electrophoresis,
place gels in the fixative enhancer
solution with gentle agitation.
Water wash steps. Decant the fixative
enhancer solution from the staining
vessel. Rinse gels in deionized or
distilled H
2
O with gentle agitation.
Decant water and replace with fresh
rinse water and rinse. Decant rinse water.
Staining step. To prepare staining
solution, add 35 ml of deionized or
distilled H
2
O to a beaker or flask with
a Teflon-coated stir bar. Add in the
following order: 5.0 ml of silver complex
solution, 5.0 ml of reduction moderator
solution, and 5.0 ml of image
development reagent. Immediately
before use, quickly add 50 ml of
development accelerator solution.
Stir well. Stain gels with gentle agitation.
Stop step. After the desired staining
is reached, place the gels in 5% acetic
acid solution to stop the staining
reaction. After stopping the reaction,
rinse the gels in high purity water for
5 min. Then the gels are ready to be
dried or photographed.
SYPRO Ruby Protein Gel Stain
Instruction manual: bulletin 4006173. Refer to
Table 11.3 for solution volumes.
Protocol
Silver Stain Plus ™ Kit
Instruction manual: bulletin LIT-442. Refer to
Table 11.4 for solution volumes and incubation times.
Components:
■ ■
Fixative enhancer concentrate
■ ■
Silver complex solution
■ ■
Reduction moderator solution
■ ■
Image development reagent
■ ■
Development accelerator reagent
■ ■
Empty 1L bottle for development accelerator reagent
Protocol
Total Protein Staining
(contd.)
1
1
4
2
2
3
5
3
4
If using the 5 L configuration, prepare
the Oriole stain solution by adding
400 ml of methanol to the 1 L bottle
of diluent. Then add 10 ml of Oriole
fluorescent gel stain concentrate
and mix well by shaking.
Note: Do not fix or wash gel prior to staining. This will
make staining less sensitive.
Place gel in a staining tray with Oriole
fluorescent gel stain. Cover the tray and
agitate for ~1.5 hr. For best results, do
not leave gel in stain for more than 2 hr.
Rinse the gel in deionized distilled
H
2
O prior to imaging. Destaining is
not necessary.
Oriole Fluorescent Gel Stain
Instruction manual: bulletin 10017295. Refer to
Table 11.2 for solution volumes.
Protocol
1
2
3