Attention: World-2DPAGE is no longer maintained. Previously submitted data can still be queried at World-2DPAGE Repository.

Silver staining protocol

1. At the end of the second dimension run, the gels were removed from the glass plates and washed in deionized water for 5 min.
2. Soaked in ethanol: acetic acid: water (40: 10: 50) for 1 hour.
3. Soaked in ethanol: acetic acid: water (5: 5: 90) for 2 hours or overnight.
4. Washed in deionized water for 5 min.
5. Soaked in a solution containing glutaraldehyde (1%) and sodium acetate (0.5 M) for 30 min.
6. Washed 3 times in deionized water for 10 min.
7. In order to obtain homogeneous dark brown staining of proteins, gels were soaked twice in a 2,7 naphtalene-disulfonic acid solution (0.05% w/v) for 30 min.
8. Rinsed 4 times in deionized water for 15 min.
9. Gels were stained in a freshly made ammoniacal silver nitrate solution for 30 minutes. To prepare 750 ml of this solution, 6 g of silver nitrate were dissolved in 30 ml of deionized water, which was slowly mixed into a solution containing 160 ml of water, 10 ml of concentrated ammonia (25%) and 1.5 ml of sodium hydroxide (10 N). A transient brown precipitate might form. After it cleared, water was added to give the final volume.
10. After staining, the gels were washed 4 times in deionized water for 4 min.
11. The images were developed in a solution containing citric acid (0.01% w/v) and formaldehyde (0.1% v/v) for 5 to 10 min.
12. When a slight background stain appeared, development was stopped with a solution containing Tris (5% w/v) and acetic acid (2% v/v).