Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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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)

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]

EDC Electrophoresis Development & Consulting, Vor dem Kreuzberg 17, 72070 Tübingen (Germany)