Silver Staining SDS Gels
· Remove the stacking gel before the first fixative step.
· Do not touch the gel near samples: handle the gel by the corners.
· Use 100 mls of volume for a mini-gel. Scale up for larger gels.
· Transfer the gel to a fresh tray where indicated. Recycle trays after rinsing them with water.
Otherwise add fresh solution after pouring off the used solution.
· Optional, for frequent silver staining: make up solutions as 10X stocks ahead of time.
1. Fix in 50% methanol, 10% acetic acid for 1 hr (to overnight) with one change fixative solution.
2. Wash the gel 3 X 20 minutes with 10% ethanol, 5% acetic acid.
3. Place gel in 100 mls ddH2O containing 0.1 g K2Cr2O7 and 20 µl of HNO3 for 6 min. Fresh tray.
4. Rinse the gel 4 X 1 minute with ddH2O. Make sure each rinse flows under the gel.
5. Place the gel in a solution of 0.1 g AgNO3 in 50 ml ddH2O. Fresh tray. Gently agitate for 15
minutes. Replace silver nitrate solution in same tray and gently agitate for 15 minutes again.
6. Rinse the gel 2 X 30 seconds with ddH2O.
7. Transfer the gel to a fresh tray containing developer solution. Make up 300 mls of developer with
9 g Na2CO3 and 150 µl 37% formaldehyde solution (formalin). [Instead of Na2CO3, 24 g of
Na2CO3(H2O)10 may be used.] Soak the gel in 100 mls of the solution for 30 seconds with vigorous
agitation. Discard the developer, and add 100 mls of fresh developer and agitate again for 30
seconds. Repeat the change of developer solution once again. Agitate gently until the desired staining