Specifications: 1L
Storage: Store at room temperature (15 ° C -25 ° C) for a period of 12 months.
Kit contents: G7210
Silver nitrate 2g
Staining solution A 100ml
Coloring solution B 100ml
Coloring solution C 100ml
Stop solution D 100ml
Note: The 1L specification refers to the volume of silver nitrate staining solution that can be prepared. The actual number of uses varies depending on the size of the PAGE gel.
Product introduction:
The principle of silver staining of nucleic acid is that silver ions can form a stable complex with nucleic acid, and then reduce the Ag+ to silver particles with a reducing agent such as formaldehyde in an alkaline environment, and the nucleic acid can be electrophoresed to be dark brown. It is mainly used for polyacrylamide gel electrophoresis dyeing. Its sensitivity is 200 times higher than EB. The whole process takes only 20 minutes. It is easy to operate and sensitive. It can detect DNA bands below 10ng.
Steps:
First, the volume of the staining solution to be prepared for the preparation of the staining solution depends on the size of the PAGE gel. The general mini-PAGE gel needs 20-30 mL. The prepared utensils are cleaned and rinsed with deionized water. The staining solution must be prepared with deionized water. If the volume of the prepared solution is not 100 mL, the amount of each component can be changed proportionally.
1, silver nitrate dye solution: weigh 0.2g silver nitrate, add to 90ml deionized water to dissolve completely, add 10ml solution A and mix, now ready to use.
2, coloring solution: Take 10ml of solution B and solution C, respectively, add 80ml of deionized water and mix well.
3, stop solution: take stop solution D 10ml plus deionized water diluted to 100ml, ready to use.
Second, dyeing:
1. After PAGE electrophoresis, transfer the PAGE gelatin to a glass plate or enamel pan and rinse twice with deionized water for 2 min each time.
2. Transfer the PAGE gel to the silver nitrate dye solution, so that the dye solution has not passed the PAGE gel and stained at room temperature for 10 min. It can be shaken slowly on a shaker to make it evenly dyed.
3. Discard the staining solution and rinse it three times with deionized water for half a minute.
4. Transfer the PAGE gel to the coloring solution, so that the coloring solution has not passed the PAGE gel, and the color is shaken at room temperature until the strip is clearly visible.
5. Optionally, transfer the PAGE gel to the stop solution and stop color development for 1 min.
6, PAGE glue after silver staining can be photographed or used for subsequent experiments.
Precautions:
1. Silver staining mainly occurs on the surface of PAGE glue, and the sensitivity can be improved by using thin glue (0.5-0.75mm).
2. For Coomassie-stained SDS-PAGE gel, rinse the gel with methanol and continue silver staining. Acetic acid will dry during the dyeing process
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Silver staining, so be sure to wash the acetic acid remaining in the PAGE gel thoroughly.
3. The response of different proteins to silver staining is different, especially the alkaline protein staining effect is poor. Therefore, it is not advisable to use silver stain to determine the ratio of different proteins.
4. Slow shaking is necessary during the dyeing process. Generally choose 40-60 rpm.
5. Cracks on the surface of the gel are mostly caused by pressure, fingerprints and surface drying, so gloves should be used throughout the process.
6. PAGE Gel background is uniform black is mostly caused by impurities in water, so the solution should be prepared using deionized water with a conductivity of less than 1 μS.
7. If a dusty or smoky gray or brown precipitate appears on the surface of the gel after dyeing, it may not be washed thoroughly during the rinsing process or if the temperature during the dyeing process is too low.
8. The deeper silver staining background is mostly caused by impurities in acrylamide.
9. PAGE Glycerin, urea, glycine, Triton X-100 and ampholytes can interfere with silver staining.
10. When operating at room temperature, temperature fluctuations often interfere with the effect of silver staining, and a constant temperature water bath can solve this problem.
11. Pretreatment with glutaraldehyde can increase the staining of various proteins by a factor of 40.
12. The glassware used for dyeing must be very clean and soaked with acid to meet the requirements.
13. Silver dye should be photographed as soon as possible, and as time goes on, the protein bands will become lighter and the background will be deeper.
14. The silver stained container is ideally a glass plate, followed by an enamel plate and a melamine plastic plate.
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