Biomedical Engineering Reference
In-Depth Information
IncubatethegelinthedarkwithSoln.Dfor1-1.5h. Remove
excess of Schiff's reagent by extensive washes with Soln. E. If the
Schiff's reagent is not removed completely, the gel becomes purple
during drying.
To visualize proteins in the presence of the red PAS staining,
incubate the gel after finishing the PAS staining with Soln. F for
1-2 h. Excess of ANS is removed by washing with Soln. E.
The protein bands fluoresce if illuminated with UV.
When no colored molar mass markers of special glycoprotein
Second staining
with ANS
markers are used, the ANS staining is recommended, because the
geometry of the gel is altered during the manipulations and com-
parison with Coomassie or otherwise stained gels is difficult.
References
Glossmann H, Neville DM Jr (1971) J Biol Chem 246:6339
2.4.4.2 Staining with Thymol
The sensitivity of staining of oligosaccharides with thymol is sim-
ilar to the PAS staining, but not so time-consuming.
A
25% isopropanol (v/v), 10% acetic acid (v/v) in ddH
2
O
Solutions/Reagents
B 0.2% (w/v) thymol (2-isopropyl-5-methylphenol) in Soln. A
C 80% (v/v) concentrated sulfuric acid, 20% (v/v) ethanol.
Caution! Corrosive!
D 30% methanol (v/v), 10% acetic acid (v/v) in H
2
O
The SDS-PAGE gel is fixed by gentle agitation in Soln. A for 1 - 2 h.
The washing is repeated with Soln. A three times, if possible one
time overnight, to remove SDS, Tris, and glycine completely.
During the subsequent incubation with Soln. B for 90 min the
gel becomes opalescent. After pouring of Soln. B, the gel is sub-
merged in Soln. C. The gel becomes clear and the glycoconjugates
appear as magenta bands. The color is stable for only a few hours.
After equilibration in Soln. D, the gel can be stained, for exam-
ple, with Coomassie.
References
Gander JE (1984) Meth Enzymol 104:447
Gerard C (1990) Meth Enzymol 182:529
2.4.5 Staining of Blotted Proteins on Membranes
2.4.5.1 Staining on Nitrocellulose with Dyes
A fast and simple, but not sensitive, reversible staining is done with
Ponceau S.
Important!
Not to be used with PVDF membranes!
Staining with Fast Green is more sensitive, but not so contrastive.
