Protein Protocols Protein Protocols

Silver Staining of Proteins 269
3.3. Staining
1. Soak the gel in the silver diamine solution for 30 min. For thick gels (1.5 mm), it is necessary
to use increased volumes so that the gels are totally immersed. Caution should be
exercised in disposal of the ammoniacal silver reagent, since it decomposes on standing
and may become explosive. The ammoniacal silver reagent should be treated with dilute
hydrochloric acid (1 N) prior to disposal.
2. Wash the gel (3 × 5 min) in water.
3.4. Development
1. Place the gel in developing solution. Proteins are visualized as dark brown zones within
10 min (see Note 6), after which the background will gradually increase (see Note 7). It
is important to note that the reaction displays inertia, and that staining will continue for
2–4 min after removal of the gel from the developing solution. Staining times in excess of
20 min usually result in an unacceptable high background (see Note 8).
2. Terminate staining by immersing the gel in stopping solution.
3. Wash the stained gel in water prior to storage or drying.
3.5. Destaining
Partial destaining of gels using Farmer’s reducing reagent is recommended for the
controlled removal of background staining that obscures proper interpretation of the
protein pattern.
1. Wash the stained gel in water for 5 min to remove the stop solution.
2. Place the gel in Farmer’s reducer for a time dependent upon the intensity of the
background.
3. Terminate destaining by returning the gel to the stop solution.
4. Notes
1. Gloves should be worn at all stages when handling gels, as silver staining will detect
keratin proteins from the skin.
2. Volumes of the solutions used at all stages should be sufficient such that the gel is totally
immersed. If the volume of solution is insufficient for total immersion, staining will be
uneven and the gel surface can dry out.
3. A mixture of alcohol, acetic acid and water (9:9:2) is recommended for gel fixation in
many published protocols, but TCA is a better general protein fixative and its use is compatible
with silver staining provided that the gel is washed well after fixation to remove
the acid.
4. In addition to removing TCA, the washing step also effectively removes reagents such as
Tris, glycine, and detergents (especially SDS) which can bind silver and result in increased
background staining.
5. Treatment of the gel with reducing agents such as glutaraldehyde prior to silver impregnation
results in an increase in staining sensitivity by increasing the speed of silver reduction
on the proteins.
6. If image development is allowed to proceed for too long, dense protein zones will become
saturated and negative staining will occur, leading to serious problems if quantitative
analysis is attempted. In addition, certain proteins stain to give yellow or red zones
regardless of protein concentration, and this effect has been linked to the posttranslational
modification of the proteins.