Biomedical Engineering Reference
In-Depth Information
2.4.2.5 Silver Staining of Glycoproteins and Polysaccharides
This procedure is not solely specific for carbohydrate side chains of
proteins. Unglycosylated proteins may also be stained. To identify
glycosylatedproteins,thesampleshouldberuninatleasttwoiden-
tical lanes: cut the gel and stain a lane with the common protein
silver stain (Protocols 2.4.2.1 to 2.4.2.4) and the other lane by the
described method. Compare pattern and intensity to identify gly-
coproteins. Glycosylated macromolecules are also stainable with
Schiff's reagent (Protocol 2.4.4.1), but with less sensitivity.
A
5% acetic acid (v/v), 40% nondenaturated ethanol (v/v) in
Solutions/Reagents
ddH
2
O
B 0.7% sodium metaperiodate (w/v) in Soln. A
C .4ml of 25% ammonia and 4.0 ml 1 N NaOH are filled up to
16.0 ml with ddH
2
Othen4.0ml 10% AgNO
3
(w/v) are added
dropwise with shaking. The precipitate formed by a drop has
to dissolve completely before giving the next. If the last drops
are not dissolved completely, some ammonia can be added
carefully. The resulting solution is pale brown. When the ad-
dition of silver is complete, ddH
2
O is filled up to 100 ml.
The solution is prepared freshly before using. After use, the
solution has to be destroyed by hydrochloric acid.
D 0.025 g citric acid, 0.185 ml of 27% formaldehyde in 100 ml
ddH
2
O
E 0.5% acetic acid (v/v) in ddH
2
O
All steps have to be done in glass vessels cleaned with concentrated
nitric acid and rinsed thoroughly with de-ionized water.
The gel is fixed after electrophoresis for at least 30 min,better
overnight, in Soln. A. The fixing solution is poured off and the gel is
agitated in Soln. B for 5 min. Wash the gel three times with ddH
2
O
for 15 min each and transfer it in a new tray after the last wash.
AtenfoldgelvolumeofSoln.Cisadded.After15min, the silver
solution is poured off and inactivated. The gel is washed three times
with ddH
2
Ofor5min each, and then it is developed with Soln. D
as described in Protocol 2.4.2.1.
References
Tsai C-M, Frasch CE (1982) Anal Biochem 119:115
2.4.2.6 Enhancement of Silver Staining
A5% l
3
(w/v) in ddH
2
O
Solutions/Reagents
B 3% oxalic acid (w/v) in ddH
2
O
C .5%
3
[Fe(CN)
6
] in ddH
2
O
D
A,B,C,andddH
2
O are mixed immediately before use in a ratio
of 1:1:1:7
After silver staining, the gel is thoroughly washed by several
changesofddH
2
O (at least four changes of ten gel volumes, 10 min
