Fast Silver Stain Kit

Instruction

The following protocol is provided for 0.75-1mm thick gel with 8 x 10cm in size. For larger gels, the solution volume in each step should be scaled up proportionally to the gel area, and for thicker gels, the incubation time for each step should also be extended correspondingly.
1. Fixation
a. After electrophoresis, place gel in 100ml of fixation solution and fix the gel for 20 minutes at room temperature on a shaker at 60-70 rpm. Fixing for more than 40 minutes or even overnight can further reduce the background.
Preparation of the fixation solution: Prepare 100ml of fixation solution by adding 50ml of ethanol and 10ml of acetic acid to 40ml of high-purity water, and mix well.
2. Gel washing with 30% ethanol
a. Discard the fixation solution and wash the gel with 100ml of 30% ethanol for 10 minutes at room temperature on a shaker at 60-70rpm.
Preparation of 30% ethanol: Prepare 100ml of 30% ethanol by adding 30ml of absolute ethanol to 70ml of high-purity water, and mix well.
3. Gel washing with high-purity water
Discard 30% ethanol and wash the gel with 100ml of high-purity water for 10 minutes at room temperature on a shaker at 60-70rpm. Longer than 10 minutes of washing helps reduce the staining background slightly.
4. Gel sensitization
Discard water and sensitize the gel with 100ml of 1X Sensitizing Solution for 2 minutes at room temperature on a shaker at 60-70rpm.
Preparation of 1X Sensitizing Solution: Prepare 1X Sensitizing Solution by adding 1ml of Sensitizing Solution (100X) to 99ml of high-purity water, and mix well. The 1X Sensitizing Solution be used within 2 hours after preparation.
5. Gel washing with high-purity water (twice)
a. Discard the 1X Sensitizing Solution and wash the gel with 200ml of high-purity water for 1 minute at room temperature on a shaker with 60-70 rpm.
b. Repeat gel washing as step a
6. Silver staining of the gel
Discard water and stain the gel in 100ml of 1X Silver Solution (1X) for 10 minutes at room temperature on a shaker at 60-70 rpm.
Preparation of 1X Silver Solution: Prepare 100 ml of 1X Silver Solution by adding 1ml of Silver Solution (100X) to 99ml of high-purity water, and mix well. The 1X Silver Solution should be used within 2 hours after preparation.
7. Gel washing with high-purity water
Discard the 1X Silver Solution and wash the gel with 100ml of high-purity water for 1-1.5 minutes at room temperature on a shaker at 60-70 rpm.
Note: Gel washing with water cannot exceed 1.5 minutes.
8. Color development
Discard the water, add 100ml of color development solution, and incubate at room temperature with agitation on a shaker until the desired degree of staining is achieved, which usually takes 3-10 minutes.
Preparation of color development solution: Prepare 100ml of color development solution by adding 20ml of Basic Developer Solution (5X) to 80ml of high-purity water, followed by the addition of 0.05ml of Development Accelerator Reagent (2000X), and mix well. The color development solution should be used within 20 minutes after preparation.
9. Termination of color development
After the desired degree of staining is achieved, remove the color development solution quickly, add 100ml of 1X Stop Solution, and incubate for 10 minutes at room temperature with agitation on a shaker at 60-70 rpm. It is normal to see bubbles, which are carbon dioxide produced during the stopping reaction.
Preparation of 1X Stop Solution: Prepare 100ml of 1X Stop Solution by adding 5ml of Stop Solution (20X) to 95ml of high-purity water, and mix well. The 1X Stop Solution should be used on the day it is prepared.
10. Gel Washing
Discard the 1X Stop Solution and wash the gel with 100ml of high-purity water for 2-5 minutes at room temperature on a shaker at 60-70 rpm.
11. Gel Storage
The stained gel can be stored in high-purity water for short-term storage or dried with appropriate methods for long-term storage.

FAQ：
1. Dark background
a. The gel is overdeveloped. The color development step should be controlled strictly, which usually takes less than 10 minutes. Overdevelopment of gel can lead to a dark background.
b. Insufficient washing. Too short time of washing, insufficient amount of washing liquid, small container used, or slow shaker results in insufficient washing. Please follow the procedures exactly as described in the manual.
c. Buffer contents in the gel were not removed completely during the fixation step. It is important to fix the gel in sufficient fixation solution for plenty of fixation time. For a gel prepared with uncommon Bis-Tris buffer, a longer fixation time is required to reduce the background.
d. Use high-quality water with a purity greater than 16 MΩ•cm.
2. Weak protein bands
a. Protein has exceptionally low or almost no cysteine residues. Cysteine residues are very important for silver staining.
b. The gel is overwashed after the silver staining step. The washing time after the silver staining step should not exceed 1.5 minutes. Otherwise, silver ions will be washed away excessively, resulting in decreased detection sensitivity.
c. Insufficient sample loading. This kit can detect BSA down to 0.3ng and its sensitivity is different for different proteins. For some proteins, more than 1ng of protein may be required to be visualized.
d. Gel washing is insufficient after the fixation step and the residual acetic acid that remains in the gel affects the subsequent reactions. Make sure the fixed gel is washed with sufficient 30% ethanol and water for plenty of time.
3. Small spots or non-protein traces on the gel
a. The gel is not covered completely by solutions. Use an appropriate container with sufficient solutions. Additionally, keep the container on a shaker at an appropriate speed to ensure an even and complete coverage of gel by the solutions.
b. The container used for silver staining is not sufficiently cleaned. The container needs to be washed thoroughly with detergent first, then with tap water, and finally washed several times with high-purity water. The container should be used exclusively for silver staining, and avoid protein contamination. To ensure adequate washing, nitric acid resistant containers, such as glassware, can be washed with 50% nitric acid after washing with detergent and tap water, and finally washed with high-purity water.
c. Fingerprints or other indentations. Always wear gloves during the operation to avoid direct contact with skin. Be careful not to squeeze, fold or rub the gel during the operation.
d. The gel is touched with metal. Non-specific marks will appear when the gel is touched with metal materials such as tweezers.
4. Smear background around 60-70 kD
The protein sample is contaminated with keratin from human skin. Wear gloves when handling the gel and keep the container lid closed whenever possible. If necessary, perform this experiment in a laminar flow hood.
5. Yellow background at the top of the gel
a. Protein samples loaded contain high DTT. Try other reducing reagents, or reduce the DTT amount in protein samples within the limit.
b. The Tris-Glycine-SDS electrophoresis system is applied. Glycine will cause a slightly yellow background at the top of the gel. Use the Tris-Tricine-SDS electrophoresis system to eliminate this yellow background.
6. Silver precipitates in staining utensils
There are residual detergents or silver staining solutions used previously. Use clean utensils only.