Encyclopedia of Experiments: Biological Techniques
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Silver Staining of Sulfated Glycosaminoglycans - ExperimentTranscript
Sulfated glycosaminoglycans, GAGs, are long, negatively charged, linear polysaccharides with repeating disaccharide unit chains sulfated at distinct positions, existing in varying lengths.
To visualize isolated sulfated GAGs of interest differing in chain length in a polyacrylamide gel following electrophoresis, carefully remove the gel from its cassette. Rinse with deionized water, removing attached gel pieces that may interfere with staining.
Treat with an acidic Alcian blue solution. Under acidic conditions, Alcian blue, a polyvalent cationic dye with a copper atom in the center, specifically interacts with negatively charged sulfated GAGs via ionic interactions, forming a deep blue water-insoluble complex.
Wash the gel with deionized water and methanol, removing any residual stain. Incubate the gel with an alkaline silver nitrate solution. Under alkaline conditions, the Alcian blue-bound sulfated GAGs provide sites for positively charged silver ions to bind and form complex.
Treat the gel in a developing solution containing formaldehyde and citric acid. Citric acid lowers local pH for optimum activity of formaldehyde, which reduces the silver ions to insoluble metallic silver within the sulfated GAG fragments, amplifying the Alcian blue signal.
Add a stop solution to lower the pH, preventing the reduction of silver ions for minimal background staining. Now, image the gel.
Alcian blue-silver stained sulfated GAG fragment bands appear dark brown or black, enabling high-sensitivity visualization and determination of the isolated GAG polymer chain lengths.
