- Protein gel from Exercise 2
- 45% (v/v) methanol + 12% (w/v) acetic acid
- 5% (v/v) methanol + 7% (w/v) acetic acid
- 10% Glutaraldehyde
- 0.01 M 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
- Remove the fixative and wash twice for 15 minutes 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 thrice 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
- 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
- Photograph or scan the gel with a densitometer, which produces 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 3-MM filter paper for autoradiography, or dried using a
commercial gel dryer.