Ag+ is reduced to Ag metal by formaldehyde at alkaline pH. It is not unreasonable to suppose that proteins bind more silver ions than the background gel. Moreover, as in photography, the deposition of metal ions is catalysed by previously deposited metal. Thus silver is deposited even more quickly around protein bands, and so they develop first. But, just like photography, the background darkens too, albeit more slowly. Therefore we have to stop the reaction at the best time to show up the weakest protein bands without letting the background become too dark.In the best cases, silver-stained gels can detect proteins two orders of magnitude below the detection limit for gels stained with Coomassie Blue.
What follows are some conjectures about the rationale for each step.
| Step | Why? |
| Ethanol Wash followed by Nitric Acid wash | Both precipitate proteins. They denature and so are not washed out
of the gel by subsequent steps.
Also both steps wash out interfering substances that could either prevent silver reduction or be stained themselves: SDS, Reducing reagents, initiators or catalysts used in gel polymerization, or buffer ions (glycine, chloride). |
| Silver Nitrate Wash | Source of Ag+ |
| Develop in formaldehyde/ Na2CO3 | Formaldehyde is oxidized to formic acid as Ag+ is reduced
to Ag(s).
Na2CO3 produces alkaline conditions. Perhaps this reduces the reaction of H3O+ with formaldehyde's carbonyl oxygen, which would protonate the latter. This would lead to formation of a geminal diol that is not as good a reducing agent as the aldehyde. Also, carbonate buffers the formic acid formed as formaldehyde is oxidized. . |
| Acetic acid wash | Acid wash drops pH and stops development. |