Silver staining protocol

1. At the end of the second dimension, the gels were washed for 5 min with Millipore filtered water (MilliQ).
2. Soaked in 40% ethanol, 10% acetic acid, 50% MilliQ for 1 h (always 200 ml per gels were used).
3. Soaked in 5% ethanol, 5% acetic acid, 90% MilliQ for at least 3 h (max. 3 days).
4. Washed in MilliQ at 4°C for 10 min.
5. Soaked in a solution containing glutaraldehyde (2%) and sodium acetate (0.5 M) at 4°C for 30 min.
6. Washed 3 times in MilliQ at 4°C for 10 min.
7. To obtain homogeneous dark brown stainig of the proteins, gels were soaked twice in a 2,7 naphthalene - disulfonic acid solution (0.05%) at 4°C for 30 min.
8. Rinsed 4 times in MilliQ at RT for 15 min.
9. Gels were stained with in a freshly made ammoniacal silver nitrate solution for 30 min. To prepare 750 ml of this solution, 6 g of silver nitrate were dissolved in 30 ml of MilliQ, which was slowly mixed into a solution containing 160 ml of MilliQ, 10 ml of concentrated ammonia (25%) and 1.5 ml of sodium hydroxide (10 N). A transient brown precipitate might form. After it cleared, MilliQ was added to give the final volume (750 ml).
10. After the staning the gels were washed 4 times in MilliQ for 4 min.
11. The images were developed in asolution containing citric acid (0.01%) and formaldehyde 37% ( 0.1% v/v) for 5 to 10 min.
12. When a slight background appeared, development was stopped with 5% acetic acid.
13. The gels were stored in MilliQ.