Silver staining protein gels (Stukenberg lab)

Protocol submitted by VGP from Stukenberg lab protocols [1]

link to protocol page [2]

Silver Staining Protocol (Not Compatible with Mass Spec)

Notes: Be careful not to touch gel during any step of the procedure with either a gloved or ungloved hand, finger etc… Use MilliQ-H2O for all steps Perform all incubations in well washed glass dishes.

1. Remove gels from glass plates in which they were run:

2. Incubate in 50% Methanol, 10% Glacial Acetic Acid, 40% H2O for 30 minutes (100 ml)

3. Incubate in 5% Methanol, 7% Acetic Acid (Glacial), 88% H2O for 30 minutes (100ml)

4. Incubate in 10% Gluteraldehyde made up in H2O from 25% stock (25 ml) for 30 minutes.

5. Wash 4 x 20-30 minutes in H2O using 200-300 ml per wash.

6. Makes 2 Solutions, A and B: A) 3 ml of 30% Ammonia Hydroxide, 0.25 g NaOH in 190 ml H2O. B) 1.2g Silver Nitrate (AgNO3) into 10 ml of H2O.

7. Add solution B to A slowly, mix gently, and add to gel, incubate for 30 minutes.

8. Make developer and stop solutions (see below).

9. Perform 5 x 1 minute washes with H2O, using 200 ml’s per wash.

10. Add developer solution (~100 ml) and wait and watch gel until you see bands come up. Quickly remove developer solution and add stop solution. (Developer Solution: 0.1g Citric Acid, 1ml 37% formaldehyde into 1L H2O. Stop Solution: 190 ml H2O and 10 ml Acetic Acid, Glacial)

11. After 10 minutes in Stop Solution wash thoroughly