Silver Staining of Polyacrylamide Gels

Use high quality nanopure water to make all of the solution and use the new petri dishes. Gently shaking the whole incubations is recommended.

  1. You can initially will gel stained with Coomassie blue and then destained with 10% acetic acid for storage. (Keep the wet gel by wrapping parafilm around the plate.)
  2. When you are ready to make your silver stain, fix blemishes by 50% methanol and 0.038% formaldehyde (formalin concentrated should be 38%). Please note formaldehyde and methanol are both toxic and should be used in a fume hood.
  3. After at least 1 hour (or overnight), aspiration of fixing solution, and wash 3-4 times with water more than 30 minutes.
  4. Meanwhile, prepare the stain. Make 5.25 ml of 0.36% sodium hydroxide. Add 350 ul of concentrated ammonium hydroxide (added in a fume hood such as ammonium hydroxide is volatile). Then, while stirring, add 200 mg / ml solution of silver nitrate before it turns completely yellow / brown. (It should take approximately 300 ul of silver nitrate.) If the solution turns yellow / brown, you have to start again. Make up to 25 ml.
  5. Change the water stains, and stain for 15 minutes.
  6. Wash for 5 minutes with water.
  7. Develop with the developer containing 125 ml of water, 0.625 ml of 10 mg / ml of citric acid, and 63 ul of 38% formaldehyde. It should take less than 10 minutes.
  8. Stop the reaction with several washes of 45% methanol, 10% acetic acid. Take pictures or dried gel. Try to keep the plate in the dark if you do not see or take a picture directly.