Silver Staining of SDS-PAGE gels
Contributed by Melissa Caimano, Ph.D. University of Connecticut Health Center Center for Microbial Pathogenesis
* DO NOT at any time touch the gel with your fingers...gloved or ungloved...Any contact with the gel with result in a dark stain where the gel was touched.
* DO NOT use the water from any of the taps..including the deionized water tap. Use only the Milli Q or distilled water.
* ONLY steps with an * may be allowed to go over the designated times. All other steps should go for only the indicated time.
* All staining should be done in GLASS (Pyrex) dishes, not plastic trays. It is better to process each gel in a separate dish since they may have different final developing times.
* The entire protocol may be shortened by reducing incubation times to 20 min each.
1. Soak gel in SOLUTION #1 for 30 min*-50ml MeOH + 10ml Acetic Acid + 40ml MQ-H2O. Can be made in advance and stored at RT.
2. Soak gel in SOLUTION #2 for 30 min*-5ml MeOH + 7ml Acetic Acid + 88ml MQ-H2O. Can be made in advance and stored at RT.
3. Soak gel in SOLUTION #3 for 30 min -20ml 25% glutaraldyhyde (Sigma G6257) + 30ml MQ-H2O. Make fresh.
4. WASH gel with MQ- H2O either 4 times for 30 min* (> 100 ml/gel/wash) OR 1 time overnight* (> 500 ml/gel/wash).
5. Soak gel for 30 min in DTT solution- 0.5 ml 100x DTT + 50ml MQ-H2O [100X DTT = 0.5 g/ml, store at -20 C]
1. Soak Gel for 30 min in SILVER SOLUTION - 0.05g Silver Nitrate (Sigma S0139) + 50ml MQ-H2O (be sure to weigh out silver using the analytical balance)
2. Prepare STOP solution- 2.42 g Citric Acid (monohydrate, Sigma C7129) + 5 ml MQ-H2O
3. Prepare DEVELOPER solution-30 ml 37% Formaldehyde (Fisher BP531) + 3g Sodium Carbonate (anhydrous, Sigma S1641) + 100 ml MQ-H2O
4. IN RAPID ORDER:
-Dump Silver Solution.
-Rinse briefly with MQ-H2O.
-Rinse gel with ~20ml Developer solution for 5 min then dump solution.
-Add remaining developer solution and watch closely for development of bands.
-Just BEFORE bands are dark enough, add the entire 5ml of STOP solution.
-When bubbling stops (~5min), dump and rinse gel with MQ-H2O.
-Gel may be stored in MQ-H2O until ready to dry but they will darken significantly if let to sit more than a few hours.