Biomedical Engineering Reference
In-Depth Information
β
well as reaction products of
-mercaptoethanol or DTT, produces
pseudobands. The artifacts made by reductants can be excluded
by using TCEP
·
HCl instead of sulfhydryl reductants (cf. Proto-
col 2.1.1).
2.4.2.2 Alkaline Development
A further variant of silver staining of proteins resulting in some
other color of protein bands has been described by Heukeshoven
and Dernick.
A
10% (v/v) acetic acid, 30% (v/v) ethanol in H
2
O
Solutions/Reagents
B .8g sodium acetate, 0.2 g Na
2
S
2
O
3
·
5H
2
O (fixing salt) dis-
solved in 60 ml ddH
2
O, then 0.5 ml 25% glutaraldehyde is
added. 30 ml of ethanol are followed and ddH
2
O is filled up to
100 ml.
µ
C
0.25% AgNO
3
(w/v) in ddH
2
O, supplemented w ith 4
l 37%
formaldehyde per 10 ml immediately before use.
D
reduction solution
:2.5%Na
2
CO
3
(w/v) in ddH
2
O, pH 11 to
11.5, supplemented with 2
µ
l 37% formaldehyde per 10 ml
immediately before use.
E 1% glycine (w/v) in ddH
2
O
After electrophoresis, the gel is fixed in Soln. A for 30 min,thenitis
equilibrated at least for 30 min or overnight in Soln. B. Next the gel
is washed with ddH
2
Othreetimesfor5min andagitatedinSoln.C
for 20 min.
Give the gel into Soln. D. After 1 min, the reducing solution is
rejected and fresh Soln. D is added to the gel. During this step the
color of bands develops. When the bands are clear visible, Soln. E
replaces Soln. D.
Washing three times with water for 5 min each and drying
finishes the staining procedure.
References
Heukeshoven J, Dernick R (1985) Electrophoresis 6:103
2.4.2.3 Silver Staining Using Tungstosilicic Acid
A
50% methanol (v/v), 10% acetic acid (v/v), 5% glycerol (v/v),
Solutions/Reagents
35% H
2
O (v/v)
B .5ml 2% AgNO
3
(w/v) in ddH
2
O and 2% NH
4
NO
3
(w/v)
in ddH
2
O are mixed immediately before use; then 2.5 ml 10%
tungstosilicic acid (w/v) in ddH
2
O, 0.25 ml 27% formaldehyde,
and 25 ml 5% anhydrous Na
2
CO
3
(w/v) in ddH
2
Oareadded
and the mixture is filled up to 50 ml.
C
5% acetic acid (v/v)
The gel is fixed with about a fivefold of its volume with Soln. A for
30 min.Thenitiswashedfourtimeswithwaterfor5min each.
