Staining Proteins in Gels

Silver staining is used for sensitive detection of proteins separated by 1D and 2D SDS-PAGE with detection limits from 0.5-5 ng based on the selectively reduction of silver ions at sites of the gel that contain proteins and other macromolecules. Many silver staining protocols and commercial staining kits are not compatible with mass spectrometry due to the use of cross-linking reagents. Commercial silver staining kits compatible with mass spectrometry are available from different suppliers. Here we describe a silver staining protocol that has been optimized for mass spectrometric analysis. The protocol results in confdent protein identifcations and high sequence coverage by MALDI MS and ESI MS due to a high recovery of peptides from the stained gel. The protocol has been tested and documented in many publications (Mortz, E et al, Proteomics 1 (11), 1359-63, 2001).

Silver stained gel

Procedure:
1. Run 1D or 2D gel. Incubate the gel in Fixer (40% ethanol, 10% acetic acid, 50% H₂O) for 1 hr.
2. Wash the gel in H₂O for at least 30 min.
Note: Overnight washing with several changes of water will remove all acetic acid, reduce background staining and increase sensitivity.
3. Sensitize the gel in 0.02% sodium thiosulfate (0.04 g Na2S2O3, 200 ml H₂O) for only 1 min.
Note: Longer time will decrease peptide recovery from the gel.
4. Wash gel in H₂O for 3 x 20 sec.
5. Incubate gel for 20 min in 4ºC cold 0.1% silver nitrate solution (0.2 g AgNO3, 200 ml H₂O, 0.02% formaldehyde, add 40μL 35% formaldehyde just before use).
Note: Staining is enhanced with cold AgNO3.
6. Wash the gel in H₂O for 3 x 20 sec.
7. Place the gel in a new staining tray.
Note: Residual AgNO3 on the gel surface and staining tray will increase background staining.
8. Wash the gel in H₂O for 1 min.
9. Develop the gel in 3% sodium carbonate (7.5 g Na2CO3 in 250 ml H₂O), 0.05% formaldehyde (add 125 μL 35% formaldehyde just before use).
Note: Change developer solution immediately when it turns yellow. Terminate when the staining is suffcient.
10. Wash the gel in H₂O for 20 sec.
11. Terminate staining in 5% acetic acid for 5 min.
12. Leave the gel at 4ºC in 1% acetic for storage. Prior to MS analysis the gel is washed in water for 3 x 10 min to ensure complete removal of acetic acid.