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Instructions are provided below for silver staining Novex® Gels using the SilverQuest™ Silver Staining Kit and the SilverXpress® Silver Staining Kit. If you are using any other silver staining kit, follow the manufacturer’s recommendations.
Materials supplied by user:
You will need following items for silver staining.
For SilverQuest™ Staining:
For SilverXpress® Staining:
For optimal silver staining results, follow these guidelines:
You will need following items for silver staining.
- Staining container
- Rotary Shaker
- Ultrapure water (>18 megohm/cm resistance recommended)
- Teflon coated stir bars
- Disposable 10 ml pipettes
- Clean glass bottles for reagent preparation
- Graduated glass cylinders
- Protein molecular weight markers (Mark 12™ Unstained Standard, recommended)
For SilverQuest™ Staining:
- SilverQuest™ Silver Staining Kit
- 30% ethanol (made with ultrapure water)
- 100% ethanol
- Fixative (40% ethanol, 10% acetic acid, made with ultrapure water)
For SilverXpress® Staining:
- SilverXpress® Silver Staining Kit
- Methanol
- Acetic acid
- Sulfosalicylic acid
- Trichloroacetic acid (TCA)
For optimal silver staining results, follow these guidelines:
- Be sure to wear rubber gloves that have been rinsed with deionized water while handling gels
- Use clean containers and designate these containers for silver staining purposes only
- Make sure the size of the container permits free movement of the gel during shaking and complete immersion in solution while staining
- Do not touch the gel with bare hands or metal objects and do not put pressure on gels while handling or changing solutions
- Use teflon coated stir bars and clean glass containers to prepare reagents
- Avoid cross contamination of kit reagents
- Use freshly made solutions
Use the reagents provided in the SilverQuest™ Silver Staining Kit to prepare the following solutions for staining:
Sensitizing solution
Ethanol 30 ml
Sensitizer 10 ml
Ultrapure water to 100 ml
Staining solution
Stainer 1 ml
Ultrapure water to 100 ml
Developing solution
Developer 10 ml
Developer enhancer 1 drop
Ultrapure water to 100 ml
Note: You may prepare all solutions immediately before starting the staining protocol or prepare them as you proceed to the next step.
Sensitizing solution
Ethanol 30 ml
Sensitizer 10 ml
Ultrapure water to 100 ml
Staining solution
Stainer 1 ml
Ultrapure water to 100 ml
Developing solution
Developer 10 ml
Developer enhancer 1 drop
Ultrapure water to 100 ml
Note: You may prepare all solutions immediately before starting the staining protocol or prepare them as you proceed to the next step.
The Fast Staining protocol (using a microwave oven) for silver staining Novex® Gels using SilverQuest™ Silver Staining Kit is described below. For the Basic Protocol and more details on the staining procedure, refer to the SilverQuest™ Silver Staining Kit Manual (IM-6070). This manual is available on our Web site at
www.invitrogen.com or contact Technical Service. For use with an 8 x 8 cm Novex® Gel, 1.0 mm thick. Use 100 ml of each solution per gel.
Note: You may have to optimize the staining protocol, if the dimensions of your gel are not the same as mentioned above.
Caution: Use caution while performing the Fast Staining Protocol using a microwave oven. Do not overheat the staining solutions.
Note: You may have to optimize the staining protocol, if the dimensions of your gel are not the same as mentioned above.
Caution: Use caution while performing the Fast Staining Protocol using a microwave oven. Do not overheat the staining solutions.
- After electrophoresis, place the gel in a clean microwaveable staining tray of the appropriate size. Rinse the gel briefly with ultrapure water.
- Place the gel in 100 ml of fixative and microwave at high power (700 watts) for 30 seconds. Remove the gel from the microwave and gently agitate it for 5 minutes at room temperature. Decant the fixative.
- Wash the gel with 100 ml of 30% ethanol in a microwave at high power for 30 seconds. Remove the gel from the microwave and gently agitate it for 5 minutes at room temperature on a rotary shaker. Decant the ethanol.
- Add 100 ml of Sensitizing solution to the washed gel. Microwave at high power for 30 seconds. Remove the gel from the microwave and place it on a rotary shaker for 2 minutes at room temperature. Decant the Sensitizing solution.
- Wash the gel twice in 100 ml ultrapure water. Microwave at high power for 30 seconds. At each wash step, remove the gel from the microwave and gently agitate it for 2 minutes at room temperature.
- Place the gel in 100 ml of Staining solution. Microwave at high power for 30 seconds. Remove the gel from the microwave and gently agitate it for 5 minutes at room temperature.
- Decant the Staining solution and wash the gel with 100 ml of ultrapure water for 20-60 seconds. Do not wash the gel for more than a minute.
- Place the gel in 100 ml of Developing solution and incubate for 5 minutes at room temperature with gentle agitation on a rotary shaker. Do not microwave.
- Once the desired band intensity is achieved, immediately add 10 ml of Stopper directly to the gel still immersed in Developing solution and gently agitate the gel for 10 minutes. The color changes from pink to clear indicating that the end of development.
- Wash the gel with 100 ml of ultrapure water for 10 minutes. If you need to destain the gel for mass spectrometry analysis, see the SilverQuest™ Silver Staining Kit Manual (IM-6070).
TOP
Prepare the reagents as described below. If you are staining two gels, double the reagent volumes. Note: The final volumes of solutions containing methanol and water reflect a volume shrinkage which occurs when these two reagents are mixed. Do not adjust volumes of components or final volume.
Fixing solution for Tris-Glycine and Tricine Gels
Methanol 100 ml
Acetic Acid 20 ml
Ultrapure water 90 ml
Sensitizing solution
Methanol 100 ml
Sensitizer 5 ml
Ultrapure water 105 ml
Staining solution
Stainer A 5 ml
Stainer B 5 ml
Ultrapure water 90 ml
Developing Solution
Developer 5 ml
Ultrapure water 95 ml
Fixing solution for Tris-Glycine and Tricine Gels
Methanol 100 ml
Acetic Acid 20 ml
Ultrapure water 90 ml
Sensitizing solution
Methanol 100 ml
Sensitizer 5 ml
Ultrapure water 105 ml
Staining solution
Stainer A 5 ml
Stainer B 5 ml
Ultrapure water 90 ml
Developing Solution
Developer 5 ml
Ultrapure water 95 ml
The following staining procedure is for 1 mm Novex® Gel. If you are using 1.5 mm Novex® Gel, double the incubation time.
Note: Gels may be stored in the second Sensitizing Solution overnight, if desired.
Note: Gels may be stored in the second Sensitizing Solution overnight, if desired.
| Step | Solution | Vol/Gel | Tricine |
1A
1B |
Fix the gel in Fixing Solution
| 200 ml N/A | 10 minutes N/A |
| 2A 2B |
Decant the Fixing Solution and incubate the gel in two changes of Sensitizing Solution.
| 100 ml 100 ml | 30 minutes 30 minutes |
| 3A 3B |
Decant the Sensitizing Solution and rinse the gel twice with ultrapure water.
| 200 ml 200 ml | 5 minutes 5 minutes |
| 4 |
Incubate the gel in Staining Solution.
| 100 ml | 15 minutes |
| 5A 5B |
Decant the Staining Solution and rinse the gel twice with ultrapure water.
| 200 ml 200 ml | 5 minutes 5 minutes |
| 6 |
Incubate the gel in Developing Solution.
| 100 ml | 3-15 minutes |
| 7 |
Add the Stopping Solution directly to the gel when the desired staining intensity is reached.
| 5 ml | 10 minutes |
| 8A 8B 8C |
Decant the Stopping Solution and wash the gel three times in ultrapure water.
| 200 ml 200 ml 200 ml | 10 minutes 10 minutes 10 minutes |