Silver Staining of Polyacrylamide Gels

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Silver Staining of Polyacrylamide Gels

Use high quality nanopure water to make all solutions and use new petri plates. Gentle shaking throughout all these incubations are recommended.

1. Your gel can initially be stained with Coomassie blue and then destained with 10% acetic acid for storage. (Keep the gel wet by wrapping parafilm around the plate.)
2. When you are ready to perform your silver stain, fix the stain with 50% methanol and 0.038% formaldehyde (concentrated formaldehyde should be 38%). Please note the formaldehyde and methanol are both toxic and should be used in the fume hood.
3. After a minimum of 1 hour (or overnight), aspirate off the fixing solution, and wash 3-4 times with water over 30 minutes.
4. Meanwhile, prepare the stain. Make 5.25 ml of 0.36% sodium hydroxide. Add 350 ul concentrated ammonium hydroxide (add in the fume hood as ammonium hydroxide is volatile). Then, while stirring, add 200 mg/ml silver nitrate just before solution turns completely yellow/brown. (Should take about 300 ul silver nitrate.) If solution turns yellow/brown, you must start again. Make up to 25 ml.
5. Replace the water with the stain, and stain for 15 minutes.
6. Wash for 5 minutes with water.
7. Develop with developer containing 125 ml water, 0.625 ml 10 mg/ml 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 a picture or dry the gel. Try to keep the plate in the dark if not viewing or taking an image right away.