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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
  1. Run SDS-PAGE gel.
  2. 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.

  3. Discard fixing solution, add 5 volumes 30% ethanol and incubate 30 minutes at RT.
  4. Repeat.
  5. Wash with 10 volumes H2O for 10 minutes at RT.
  6. Repeat.
  7. 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.
  8. Discard solution and rinse both sides of gel for ~20 seconds each under stream of ddH2O.

    Be careful in handling the gel.

  9. 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.

  10. 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.

© 2014 The University of Texas MD Anderson Cancer Center