Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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Because silver staining is very sensitive for detection (picograms!), this is also valid for disturbing  influences, which can be introduced by, for instance, differing qualities of H₂OBidist.
Because sodium thiosulfate interferes with the reduction of formaldehyde - as it complexes Ag+ -, the following procedure should be  performed:

Ammoniacal silver staining (IEF of IgG)

Pipet first 100 ul of the complete silvering solution in a weighting tray.
Then pipet in this droplet 100 ul of the complete developer.
The colour of the droplet should turn from transparent to yellow-green-brown within 3 seconds

Alkaline silver staining (SDS, IEF, 2D)

Adjusting the amount of sodium thiosulfate in the developer:
Prepare the silver solution, and bath gel in it.

General Test

During the silver step of the gel, prepare the developer, but without sodium thiosulfate. Pipet 50 uL of the silver solution in a weighing tray.

Pipet 50 uL of the developer into the silver solution droplet and count to 3. Within these 3 seconds the color of the droplet should turn into brown-black.

This is an important check, whether all substances are present in the solutions: AgNO₃,
Na₂CO₃, and formaldehyde, all three substances must be present, otherwise no success!).

If this test takes longer than 10 seconds, check quality of chemicals and cleanness of the equipment (staining trays).

 Important: from the silvering step on, no plastic trays should be used, but only glass or stainless steel (some plastic trays contain softeners, which interfere with the silver staining procedure!)

Optimization Tests (DNA Silver-Staining)

Add 25% of the above recipes amount of sodium thiosulfate to the developer, and pipet 50 ul developer on 50 ul silver solution. If no change can be observed, add another 25%  until the following happens:

Do not forget: rinse the tray and the gel (on both sides) thoroughly between the silvering and the development step.
Control: Important: To be able to  discriminate between sample disease and electrophoretic mistakes, and to determine the silver staining effectivity there should run a marker lane on every gel. The 100-basepair ladder (Amersham Pharmacia 27-4001-01) - diluted  10 ul marker + 1290 ul sample buffer - application of 6.5 ul, must be well visible

Optimization Tests (Protein Silver-Staining)

The droplet should turn brown immediately
 and turn black within ca.5 - 10 seconds, if not:
Raise the formaldehyde-concentration in the Silvering and the  develloping steps. For the first trial raise about 25%

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