Exercise 4.4 - Silver Staining of Gels
Figure 4.8 Silver stained protein gel
- Protein gel from Exercise 4.2
- 45% (v/v) Methanol + 12% (w/v) acetic acid
- 5% (v/v) Methanol + 7% (w/v) acetic acid
- 10% Glutaraldehyde
- 0.01M Dithiothreitol
- Silver nitrate solution
- Sodium citrate / formaldehyde
- Kodak Farmer's Reducer or Kodak Rapid Fixer
- Fix gels by gently rocking them in a solution of 45% methanol / 12% acetic acid until the gels are completely submerged. Fix for 30 minutes at room temperature.
- Remove the fixative and wash 2x for 15' each with 5% ethanol / 7% acetic acid. (Gels thicker than 1 mm require longer washing.)
- Soak the gels for 30 minutes in 10% glutaraldehyde.
- Wash 3x with deionized water, 10 minutes each.
- Place in dithiothreitol for 30 minutes.
- Place in silver nitrate solution for 30 minutes.
- Wash for 1 minute with deionized water.
Dispose of used silver nitrate solution immediately with continuous flushing. This solution is potentially explosive when crystals form upon drying.
- Place in sodium citrate / formaldehyde solution for 1 minute.
- Replace the sodium carbonate/formaldehyde solution with a fresh batch, place gels on a light box and observe the development of the bands. Continue to rock gently as the gel develops.
- When the desired degree of banding is observed (and before the entire gel turns black), withdraw the citrate / formaldehyde solution and immediately add 1% glacial acetic acid for 5 minutes.
- Replace the glacial acetic acid with Farmer's reducer or Kodak Rapid Fixer for 1 minute. Remove Farmer's reducer and wash with several changes of deionized water.
- Photograph or scan the gel with a densitometer. Figure 4.8 demonstrates a typical silver stained protein gel.
- For storage soak the gel in 3% glycerol for 5 minutes and dry between dialysis membranes under reduced pressure at 80-82° C for 3 hours. Alternatively, place the wet gel into a plastic container (a storage bag will do) and store at room temperature. If desired, the gels may be dried between Whatman 3MM filter paper for autoradiography, or dried using a commercial gel dryer.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org