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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

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Non-Kit based Silver staining Method:

Hahn Lab: Kyle Coachman, Jeff Ranish, Steve Hahn

last modified 5/13/02

Materials Needed:

Silver Nitrate 1% Citric Acid: 100 ml of DI water + 1 g of Citric Acid 30 % NaOH(7.5 M): 100 ml of DI water + 30 g of NaOH 14.8 M Ammonium Hydroxide 38% Formaldehyde Ultra-pure water, use this for all steps and reagents 50% Aqueous Glutaraldehyde (optional) 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

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?Method:

Make 7% Acetic Acid: 186 ml of water + 14 ml of acetic acid 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). Soak gel in 7% acetic acid for 7 minutes. Soak gel in 200 ml of 50% methanol for 20 minutes. Soak gel in 200 ml of 50% methanol for 20 minutes. Prepare Solution A: 0.8 g of silver nitrate + 4 ml of water Rinse gel in ~200 ml water for 10 minutes 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.

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 To make staining solution: add solution A to solution B dropwise while stirring then add 76 ml of water. Soak gel in the staining solution for 15 minutes Rinse gel in ~200 ml water for 5 minutes Rinse gel in ~200 ml water for 5 minutes Make developing solution: 200 ml of water + 1 ml of 1% citric acid + 100 ul of 37% formaldehyde Soak gel in developing solution until bands are visible usually 2 to 15 minutes 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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