The proteins are separated on one-dimensional SDS-PAGE, silver stained and analyzed by mass spectrometry. We used a modified silver staining protocol from Shevchenko et al.1.

Silver staining:

All steps are performed on a shaking table at room temperature except step 5.

1. Fix gel with destaining solution

 - methanol: acetic acid : water (40 : 10 : 50)

 - time: 20 - 30 min

2. Rinse gel with water to remove acid

 - several times

 - time: 3 - 4 hours

3. Sensitize gel with 0.02% (w/v) sodium thiosulfate

 - 0.1g sodium thiosulfate in 0.5L water

 - time: 1 - 2 min

4. Rinse gel with two changes of water (1 min each

5. Incubate gel in (chilled ) 0.1% (w/v) silver solution

 - 0.5g silver nitrate in 0.5L water

 - time: 20 - 40 min, incubate at 4°C

6. Rinse gel with two changes of water (1min each)

7. Develop gel with developing solution (0.04% (v/v) formaldehyde, 2% (w/v) sodium carbonate)

 - 10g sodium carbonate in 0.5L water

 - 540µl formaldehyde (37%) in 0.5L water

 - on a shaking table

 - replace solution when it turns yellow

8. Quench development when sufficient staining is obtained

 - add 1% acetic acid

 - 5 ml acetic acid in 0.5L water

9. Store the silver stained gel in 1% (v/v) acetic acid solution at 4°C

Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996). Mass Spectrometric Sequencing of Proteins from Silver - Stained Polyacrylamide Gels. Analytical Chemistry 68, 850 - 858.