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Dr. Hanspeter Schickle
Wolfgang Gstrein

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 General Silver Staining (Strategy of Optimization)

[SDS Silver]
[IEF Silver]
[Droplet Test]
[Trouble Shooting AG]


The pinciple of silver staining is the following:

In the gel the soluble, non visible Ag+ -ions are reduced
 to the metallic, black, and visible Ag0.

The Ag+ donor is: AgNO3

Oktober 18, 2021

At the same time, the redox potential has to be chosen in that way, that the Ag+ ion, which is complexed in the polyacrylamide gel, can only react in the presence of an additional compound: a  biomolecule like a protein or DNA.

The reducing solution: 0.037 % formaldehyde, and pH 12 due to 2.5 % sodium carbonate

To avoid re-desolving of precipitating Ag+-ions (brown, visible), such as AgCl, use:

Ag+ -complexing compound: 0.002 % sodium thiosulfate (only DNA silver-staining)

silver-bad silver-good

Solutions and Procedure (General, DNA-Silver-Staining)

A Fixing and Washing

At the beginning, the gels have to be fixed and washed. Different recipes for proteins and DNA.

Fixing DNA-gels, old recipe: 15 % ethanol / 5 acetic acid with the gel swimming on the liquid, gel side looking downward. 45 min at room temperature, 20 min when preheated to 50°C  Washing with Bidest, gel swimming on the surface looking downward: 3 times 13 min at room temperature, 3 times 5 min when preheated to 50°C

New and better recipe:
Fixing with 0.6% Benzenesulfonic acid (= Phenyl sulfonic Acid) [Merck 8.00468.0250] in 24% Ethanol

B  The Silver Solution (prepare freshly)

 0.1% AgNO3 [Merck 1.01512.0100] brings the Ag+ ions into the gel; stock solution: 2% (w/v).
Formaldehyd is the reducing reagent, but does not yet work, because the pH value is slightly acidic.
The actual solution: 10 ml silver solution + 200 ul formaldehyde (37%) [Merck 1.04003.1000] on 200 ml with H2O Bidest.

During the silvering, the gel lays at the bottom of the tray, gel side looks upward! 45 min at room temperatur, 20 min when preheated to 40°C

 C The Developer (prepare freshly):

2.5 % Na2CO3 [Merck 1.06392.1000] shifts the pH to 12, fast start, because formaldehyde is already present (silver solution!). Na2CO3 - stock solution: 10% (w/v).
0.037% formaldehyde reduces - together with the biomolecules - the Ag+ -ions).
0.002% sodium thiosulfate [Merck 1.06516.0500] complexes precipitating Ag+ -ions (brown background!); recommended stock solution: 2% (w/v), keep cool for only 1 week.
The actual solution: 50 ml Na2CO3 + 200 u l Formaldehyd + 150-200 ul Na-thiosulfate per 200 ml.

During the developing step, the gel lays at the bottom of the tray, gel side looks upward!3-6 min at room temperature, dont heat! Best results when precooled to 8°C!

Testing and optimization of the 2 imortant solutions: The droplet-test”.

D Stopping, Preserving and Drying

At the end, the gels have to be desilvered, preserved and dried.

This can be done in one solution: 2% Glycin + 0.5 EDTA-Na. 20 min at room temperature. Air dry overnight.

E Ammoniacal Silver Staining, additional chemicals

Ammonia 25% [Merck 1.105432.1000], Citric Acid [Merck 1.00244.0500]

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