Silver Staining of Gels

Materials

• 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

Procedure

1. 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.

    

2. Remove the fixative and wash 2x for 15'' each with 5% ethanol / 7% acetic acid. (Gels thicker than 1 mm require longer washing.)

    

3. Soak the gels for 30 minutes in 10% glutaraldehyde.

    

4. Wash 3x with deionized water, 10 minutes each.

    

5. Place in dithiothreitol for 30 minutes.

    

6. Place in silver nitrate solution for 30 minutes.

    

7. 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.

    

8. Place in sodium citrate / formaldehyde solution for 1 minute.

    

9. 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.

    

10. 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.

     

11. 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.

     

12. Photograph or scan the gel with a densitometer. Figure 4.8 demonstrates a typical silver stained protein gel.

     

13. 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.