Silver Stain SDS-PAGE Gel for Protein
Notes:
- This method is 100 to 1000 times more sensitive that Coomassie
- You can detect as little as 0.1-1 ng protein
- Wear gloves and handle gels carefully, since pressing on the gel can cause staining artifacts
- Use clean glassware and ddH2O
- Run SDS-PAGE gel.
- Fix proteins in gel by incubating 4-12 hours at RT, shaking gently in ethanol:glacial acetic acid:H2O (30:10:60) with at least 5 gel volumes of solution.
Be wary of fumes. - Discard fixing solution, add 5 volumes 30% ethanol and incubate 30 minutes at RT.
- Repeat.
- Wash with 10 volumes H2O for 10 minutes at RT.
- Repeat.
- Wearing gloves, add 5 volumes 0.1% AgNO3 (freshly diluted from 20% stock, which is stored in a dark container). Incubate 30 minutes at RT.
- Discard solution and rinse both sides of gel for ~20 seconds each under stream of ddH2O.
Be careful in handling the gel. - Add 5 volumes freshly made 2.5% sodium bicarbonate/0.02% formaldehyde. Incubate at RT.
Formaldehyde solution is generally provided as a 37% stock solution.
Watch carefully. The stained bands should appear within a few minutes.
While watching the gel, be wary of the formaldehyde fumes!! These are toxic!!
If you overdevelop the gel, it will begin to turn yellowish-brown all over. - Quench the reaction by washing the gel in 1% acetic acid for a few minutes -- again, be wary of fumes. Then wash the gel for several 10-minute periods in H2O.