Biol 307

Silver staining of protein gels

All steps are done for 30 min with gentle agitation unless noted otherwise. Prepare 100 ml for 4 BioRad minigels.

1. 50% methanol, 10% acetic acid

2. 5% methanol, 7% acetic acid.

3. 10% gluteraldehyde.

4. 4 X 30 min in water or overnight

5. 5 µg/ml DTT.

6. Pour off solution and without rinsing add 0.1% silver nitrate.

7. Rinse once rapidly with 20 ml water and once rapidly with 20 ml developer (50 µl of 37% formaldehyde in 100 ml 3% sodium carbonate, mixed just before use).

8. Incubate with 50 ml developer until desired level of staining is obtained. Stop staining by adding 5 ml 2.3M citric acid/100 ml developer solution (=2.5 ml 2.3M citric acid/50 ml developer solution). Agitate for 10 min.

9. Wash with water 3 X 10 min.

10. Photograph.

11. To store, soak in 0,03% sodium carbonate, seal in bag or cellophane.

Reference: Morrissey, J.H. Anal Biochem 117, 307-310 (1981).