Bio-Rad Silver Stains - Manual (Page 4)

fate pentahydrate in 1 liter deionized water. Prepare the destain by combining
equal parts of the two solutions immediately prior to use. Remove the gel from the
destain before complete destaining has occurred. Wash several times in deionized
water to remove the destain and prevent fogging. Stop the destaining process with
10% acetic acid for 15 minutes. Wash the gel for at least 1 hour with several
changes of water. To restain, begin with step 8, Table 1.
An alternative destain is Kodak rapid fix, solution A. Add 12 ml of rapid fix
to 88 ml deionized water to prepare the destain. Stop the destaining process with
2.4 g Kodak hypo clearing agent dissolved in 100 ml deionized water, for 15 min-
utes. Wash the gel for at least 1 hour with several changes of water. To restain,
begin with step 8, Table 1.
3.7 Storage of Stained Gels
After staining is complete, gels can be stored indefinitely in zip-lock plastic bags
with just a few drops of water. Alternatively, gels may be dried on filter paper.
Change the stop solution two or three times (at least 5 minutes for each step) to
remove all of the developer before drying. This will prevent continued development.
Drying gels between two pieces of cellophane also eliminates the problem of the
gel darkening upon drying.
3.8 Photographing Gels
Gels may be photographed during development or after stopping with 5%
acetic acid. Photographing should be done on a bright light box, however, devel-
opment may be accelerated if it has not yet been stopped. If the staining contain-
er is not optically clear, the gel should be placed on a glass plate or directly on the
light box for photographing.
Suggested Settings
Camera Polaroid-type 35 mm (50 mm lens)
(Polaroid Model MP-4)
Film Type 667, positive Kodak panatomic-X, asa 32
F stop F32 F16
Speed 1/125 sec. automatic setting
Height 1-2 feet 1-2 feet (on tripod)
3
3.2 Gel Handling
Wear rubber gloves that have been washed and rinsed with deionized water. Use
glass containers and make sure that the volumes are sufficient to allow free move-
ment of the gel during shaking. An orbital shaker is recommended to promote
mixing during all steps. Use a separate dish for each gel. Do not put pressure on
gels during handling or when decanting solutions.
3.3 Temperature
The oxidizer, silver reagent, and developer solutions should be at room tem-
perature (23-25 ˚C) for use. If heating is necessary, dilute the oxidizer and silver
reagent before warming to 25 ˚C. Use immediately. The developer can be heated
to 50 ˚C to enhance development (see “Band Development” below).
3.4 Convenient Stopping Point
Protein gels can be stored indefinitely in 40% methanol/10% acetic acid prior
to staining.
3.5 Band Development
Bands usually appear dark brown against a pale background. The duration of
the development steps is very approximate, and development should be monitored
closely. The third volume of developer may not be necessary, especially when a water
wash is used between the silver reagent and developer steps.
Stop development when the bands reach the desired intensity in relation to
background. If drying the gel on filter paper, stopping development just before
the desired intensity is reached will help keep the gel from turning darker on the
filter paper (see Troubleshooting Guide). The rate of development is highly tem-
perature dependent. Developer can be heated to 50 ˚C if faster development is
desired.
3.6 Destaining
Gels allowed to develop too long will have high background or surface deposits
of silver (mirroring). Destaining can be performed using a photographic reducer
such as the following, which was developed by Switzer, et al.
3
Dissolve 37 g of sodium chloride and 37 g of cupric sulfate anhydrous in 850
ml of deionized water. Add concentrated ammonium hydroxide until the precipi-
tate that forms is completely dissolved to give a deep blue solution. Adjust to 1 liter
with deionized water. Prepare a second solution containing 436 g sodium thiosul-
2
LIT34B 6/17/98 2:04 PM Page 2