Silver staining is more sensitive than ethidium bromide for double-stranded DNA and detects single-stranded DNA or RNA with no loss of sensitivity. Silver staining relies on the reduction of silver cations to insoluble silver metal by nucleic acids. This chemical reaction is insensitive to the macrostructure of the DNA molecule. Reduced silver grains deposit in the gel around the DNA bands, creating a “latent image”. The latent image is developed for visibility by soaking the gel in a solution of silver cations and a reducing agent. The silver granules in the latent image catalyze the further reduction and deposition of silver from the solution. Bands manifest as dark brown or black regions that appear before significant background develops. Development is stopped by altering the pH of the gel to a point where silver reduction is no longer favored.

The mechanism whereby nucleic acids reduce silver is not well defined. Staining is enhanced by treatment with oxidants, and it may be that such treatment oxidizes vicinal sugar diols to highly reductive aldehydes, which are known to reduce silver (e.g. Tollens test). However, if this were the primary mechanism, silver staining would be expected to destroy the DNA. Recent reports indicate that enough DNA survives silver staining to provide a template for PCR amplification. This may reflect either the nondestructive nature of silver staining or the extreme sensitivity and power of the PCR reaction.

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Silver Staining with the Sterling Silver Kit

For mini-gels (10X7cm), use 100ml of each solution. For larger gels, increase STERLING volumes appropriately to immerse gel to a depth of 1cm. Wash mini-gels in 200ml volumes of water, and agitate continuously during all steps. Glassware must be clean, and the water should be distilled or high-quality deionized.

FIX

1. Incubate the gel for 25 minutes in 100ml of the standard mixture of 5:5:1 methanol:water: acetic acid.
2. Decant fixative, then add reconstituted STERLING Fixative (45ml water, 50ml methanol, 5ml STERLING Fixative Concentrate) and fix for an additional 5 minutes.

WASH

1. Rinse the gel twice for 15 minutes in deionized water. The addition of 0.1% nonionic surfactant will aid in submerging the gel.
2. While the gel is washing, prepare the Staining Solution (see below). Do not combine the two-component solutions until just prior to use.PREPARATION OF STAINING SOLUTION
   • Dilute 25ml Reagent A with 25ml of water.
   • Dissolve 2.8 grams of Reagent B in 50ml of water. STIR UNTIL COMPLETELY DISSOLVED (approx. 5-10 minutes).
   • Immediately prior to use, pour the solution from Step 1 into the solution from Step 2 while stirring, and pour over gel. The combined solution has a useful life of 20 - 30 minutes.

STAIN

1. Decant wash solution and immerse gel in combined staining solution.
2. Bands will begin to appear in 5-10 minutes. When desired intensity is achieved, stop development by immersing the gel in a 5% acetic acid solution.

 

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