Biomedical Engineering Reference
In-Depth Information
each). Then the gel is slightly agitated in Soln. D for 0.5 to 3 min and
is washed again with ddH
2
O. During this manipulation the color
of the brown to black bands shift to blue, which is mostly better
to recognize. Vanishing of different colors of individual bands is
adisadvantageofthisprocedure.
References
Poehling HM, Neuhoff V (1981) Electrophoresis 2:141
Berson G (1983) Anal Biochem 134:230
Heukeshoven J, Dernick R (1985) Electrophoresis 6:103
2.4.2.7 Reducing of Silver-Stained Gels
Reducing (weakening) of overdyed gels should be the ultima ratio,
because it is accompanied by a loss of information. Because the
principle of silver staining is the same as in (silver halogenide)
photography, reduction of dark background of a gel or to dark
bands by oxidation of colloidal silver is done with a photographic
reducer.
A
15% acidic fixing salt (Na
2
S
2
O
3
ยท
5H
2
O) (w/v), 1.2% thiourea
Solutions/Reagents
(w/v) in ddH
2
O
B5%
3
[Fe(CN)
6
] (w/v) in ddH
2
O
Solutions A and B are mixed immediately before use in a ratio given
in Table 2.15.
The weakening or destaining is performed under visual control
and finished by incubation with ddH
2
O.
After extensive washings with ddH
2
O(gelswithexceptionof
remaining bands and washings are absolutely colorless), the silver
staining can be repeated or a further identification method is used.
Table 2.15.
F
ARMER
's reducer
Reducing power
Volume parts
AB
2
O
Fast and intensive
1
1
2
Medium
1
1
4
2.4.3 Copper Staining of SDS-PAGE Gels
A4%
l
2
(w/v) in ddH
2
O
Solution/Reagent
After electrophoresis, the gel is submerged without fixing into
Soln. A (about 10 ml per milliliter of gel) for 5 min with gentle
agitation. The gel is washed with ddH
2
Ofor2-3min.Theprotein
bands appear clear, whereas the rest is milky opalescent. This effect
is best visible using a black background.
