1.3.4. Staining gels with silver salts

Various methods have been developed for staining polypeptides with silver salts after separation by SDS-PAGE, including more or less laborious and more or less sensitive methods. The procedure for silver staining of proteins in polyacrylamide gels described below is based on that described by Blum et al. (1987). A polyacrylamide gel with dimensions 100 x 120 x 0.9 mm requires a volume of at least 200 ml of all solutions. The plastic container used should be adapted to the size of the gels to allow complete immersion. Solutions containing thiosulfate have to be prepared freshly to obtain sensitive and reproducible staining results. Powder-free disposable gloves should be worn when handling/transferring gels, as silver stain will detect proteins and oil transferred from fingers.

1. Prepare a fixative solution containing

   1.1. 50 % (v/v) methanol.
   1.2. 12 % (v/v) acetic acid.
   1.3. 0.5 ml of 37 % formaldehyde.
   1.4. Dilute with ddH2O to 1 l.

2. Incubate gel in the fixing solution for 30 min at room temperature with gentle shaking.

3. Wash for 10 min at room temperature in aqueous solution containing 50% (v/v) ethanol, with gentle shaking.

4. Repeat step 3.

5. Incubate gel for 5 min in a solution containing Na2S2O3·5H2O (0.2 g/l).

6. Rinse gel in water for 20 s.

7. Repeat step 6 twice.

8. Incubate for 10 min at room temperature with gentle shaking in a solution containing:

   8.1. AgNO3 (2 g/l),
   8.2. 37% formaldehyde (0.75 ml/l).

9. Rinse the gel in water for 20 s.

10. Repeat step 9.

11. Add developer solution, made up from:

   11.1. Na2CO3 (60 g/l),
   11.2. 37% formaldehyde (0.5 ml/l),
   11.3. Na2S2O3·5H2O (4 mg/l).

12. Incubate gel at room temperature with gentle agitation and carefully watch the developing process.

13. Stop developing process once the desired band intensity and contrast are obtained by adding a solution containing

   13.1. 50 % (v/v) methanol.
   13.2. 12 % (v/v) acetic acid.
   13.3. 38 % ddH2O.

14. Wait for a few minutes, then wash the gel with ddH2O.

15. Preserve the gel by drying or in wet condition within a sealed plastic container; add a few drops of glycerol to keep the gel soft.