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| This protocol describes a method for silver staining a polyacramide protein gel in order to visualize the proteins in the gel. The silver ions bind to the side chains of the amino acids and are subsequently reduced to metallic silver by the development solution. It is possible to detect as little as 0.1 - 1 ng of protein with silver stains. This method is 100 to 1000 times more sensitive than Coomassie Blue staining. |
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1. Remove the gel from the glass plates with a spatula or razor blade (see Hint #1).
2. Transfer to a large plastic or glass container (see Hint #2).
3. Completely cover the gel in Fixing Solution #1.
4. Incubate at room temperature for at least 1 hour on a rocking platform (see Hint #3).
5. Decant the Fixing Solution (see Hint #4).
6. Completely cover the gel in 50% Ethanol.
7. Incubate at room temperature for approximately 20 min on a rocking platform.
8. Decant the 50% Ethanol.
9. Repeat the incubation in 50% Ethanol (Steps #6 to #8) 3 more times.
10. Completely cover the gel in Thiosulfate Solution and incubate at room temperature on a rocking platform for no more than 1 minute (see Hint #5). Promptly pour off the solution.
11. Quickly wash the gel using three 20 sec washes of ddH2O.
12. Completely cover the gel in Silver Nitrate Solution.
13. Incubate for 20 minutes on a rocking platform.
14. Discard the Silver Nitrate Solution.
15. Wash the gel 2 times using ddH2O (approximately 20 sec per wash).
16. Completely cover the gel in Developing Solution.
17. Incubate at room temperature until the silver stain has developed to the desired darkness (see Hint #6)
18. Discard the Developing Solution.
19. Wash the gel 2 times using ddH2O (approximately 20 sec per wash).
20. Completely cover the gel in Fix Solution #1 to stop the development.
21. Incubate at room temperature on a rocking platform for 10 min.
22. Discard the Fix Solution #1.
23. Completely cover the gel in Glycerol Solution.
24. Incubate at room temperature for 1 to 2 hours (or overnight).
25. Dry the gel in gel dryer and seal in plastic wrap.
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| Silver Nitrate Solution |
| 187 ml 37% (v/v) Formaldehyde (CAUTION See Hint #7)
Prepare just before use
2 mg/ml Silver Nitrate
250 ml H2O
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| Thiosulfate Solution |
| 0.2 mg/ml Thiosulfate
Prepare just before use
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| 50% (v/v) Ethanol |
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| Fixing Solution #1 |
| 10% (v/v) Acetic Acid
50% (v/v) Methanol
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| Glycerol Solution |
| 5% (v/v) Glycerol
20% (v/v) Ethanol
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| Developing Solution |
| 30 g Sodium Carbonate
Prepare just before use
250 37% (v/v) Formaldehyde (CAUTION See Hint #7)
Bring volume of 500 ml using ddH2O
10 ml Thiosulfate Solution
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Silver Nitrate
Methanol
Ethanol
Formaldehyde
Acetic Acid
Sodium Carbonate
Glycerol
Thiosulfate
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1. Be careful to not distort the gel and to clearly mark a gel corner so that you will know the order of the samples that were loaded onto the gel. Wear gloves when handling the gel.
2. If using plastic, line the plastic container with Saran wrap to prevent the gel from sticking to the plastic surface. Never touch the gel with your bare hands because this will contaminate the gel surface with proteins.
3. This step fixes the proteins to the Acrylamide in the gel. After fixing, proteins will not be washed away or diffuse during the protein staining process.
4. Make sure to hold the gel down with light pressure using a new pair of gloves.
5. Do not expose the gel to Thiosulfate Solution for more than 1 minute.
6. Pay attention to the appearance of the brown protein bands; leave the gel in the developing solution until the protein bands are stained to the desired intensity.
7. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
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