蛋白质电泳技术-2
Decrease toxicity of destain even more!
The destain procedure can be made even less toxic by replacing the destaining solution completely with MilliQ water and no organic solvents. Basically, a freshly stained gel is immersed in water then microwaved on high for 15 min for a 0.75 mm thick gel or 25 min for 1.5 mm thick gel.
This method and others are available from Elsevier Trends Technical Tips Online (http://tto.trends.com/tto/index_of_TTO.shtml) under the Electrophoresis section. Reference no. is T01089.
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Silver Staining of SDS-PAGE gels
IMPORTANT TIPS:
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.
GEL PREPARATION:
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.
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.
Soak gel in SOLUTION #3 for 30 min -20ml 25% glutaraldyhyde (Sigma G6257) + 30ml MQ-H2O. Make fresh.
WASH gel with MQ- H2O either 4 times for 30 min* (> 100 ml/gel/wash) OR 1 time overnight* (>500 ml/gel/wash).
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]
GEL STAINING:
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)
Prepare STOP solution- 2.42 g Citric Acid (monohydrate, Sigma C7129) + 5 ml MQ-H2O
Prepare DEVELOPER solution-30 ml 37% Formaldehyde (Fisher BP531) + 3g Sodium Carbonate (anhydrous, Sigma S1641) + 100 ml MQ-H2O
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.
