Silver Staining SDS PAGE Gels

We currently use the SilverQuest Silver stain kit from Invitrogen. This method is faster than the method listed below. The rapid method works well for most applications. Click here for instructions to the SilverQuest kit


Non-Kit based Silver staining Method:

Hahn Lab: Kyle Coachman, Jeff Ranish, Steve Hahn

last modified 5/13/02

Materials Needed:

  1. Silver Nitrate
  2. 1% Citric Acid: 100 ml of DI water + 1 g of Citric Acid
  3. 30 % NaOH(7.5 M): 100 ml of DI water + 30 g of NaOH
  4. 14.8 M Ammonium Hydroxide
  5. 38% Formaldehyde
  6. Ultra-pure water, use this for all steps and reagents
  7. 50% Aqueous Glutaraldehyde (optional)
  8. Glass tray or Novex Stain Ease Gel tray. If using glass, make sure to clean well with soap and DI water. If using Novex tray, use only trays designated for use with silver staining – do not use trays which have been used for coomassie staining

 

 Method:

  1. Make 7% Acetic Acid: 186 ml of water + 14 ml of acetic acid
  2. Make 50% Methanol: 200 ml of water + 200 ml of methanol. Optional – for extra fixation/crosslinking add 240 µl of 50% glutaraldehyde to the 50% methanol (makes solution 0.03% glutaraldehyde).
  3. Soak gel in 7% acetic acid for 7 minutes.
  4. Soak gel in 200 ml of 50% methanol for 20 minutes.
  5. Soak gel in 200 ml of 50% methanol for 20 minutes.
  6. Prepare Solution A: 0.8 g of silver nitrate + 4 ml of water
  7. Rinse gel in ~200 ml water for 10 minutes
  8. Rinse gel in ~200 ml water for 10 minutes

    Note: Steps 7 and 8 are very important for the NuPAGE gels if you skip these steps or do not rinse the gel for long enough the gel will develop too quickly and have significantly more background.

  9. 5 minutes before end of final water rinse prepare solution B: 21 ml of water + 250 ul of 30% NaOH (to make 0.36%) + 1.4 ml of 14.8M ammonium hydroxide
  10. To make staining solution: add solution A to solution B dropwise while stirring then add 76 ml of water.
  11. Soak gel in the staining solution for 15 minutes
  12. Rinse gel in ~200 ml water for 5 minutes
  13. Rinse gel in ~200 ml water for 5 minutes
  14. Make developing solution: 200 ml of water + 1 ml of 1% citric acid + 100 ul of 37% formaldehyde
  15. Soak gel in developing solution until bands are visible usually 2 to 15 minutes
  16. Stop development by rinsing gel with 3 changes of ~200 ml water

 The sensitivity of this method should be in the 10ng/band range.

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