Silver stain protocols
These procedures are compatible with mass spectrometry analysis of
proteins. Quantities are for large (20 cm x 20 cm) gels.
For an example of a stained gel, click here. (135 µg of
E. coli protein was run on a pH 4-7 IEF strip,
then run on a 22 cm x 22cm gel.)
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Vorum |
Desiderio Silver destain to be used when restaining desired. |
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Fix 2-18 hrs. (50% MeOH/12% acetic acid, 0.05% formalin), 1 liter/gel Wash three times in 35% EtOH/water for 20 min Wash twice in distilled water for 10 minutes Sensitize 2 min with 0.5L of 100 mM sodium thiosulfate, 30 mM potassium ferricyanide Wash in 0.5 L water for 10 min. four times Stain 20 min. with 1 L 0.2% silver nitrate, 0.076% formalin Wash in water 1 min. twice Develop till dark enough with 1 L of 6% sodium carbonate, 0.05% formalin, 0.0004% sodium thiosulfate Stop 5 min. with 1 L of 50% MeOH/12% acetic acid Store in 1% acetic acid/water
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Water 5 min (3X) Destain whole gel 15 min. with 1:1 v/v 30 mM potassium ferricyanide and 100 mM sodium thiosulfate prepared fresh Water 5 min. (10X) Either proceed to fix step for staining or store in 1% acetic acid/water
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References: Vorum and Blum procedures: presented at 48th American
Society for Mass Spectrometry Conference on Mass Spectrometry June
11-15 2000, Long Beach, CA, poster TPE 191. Now described in:
Lin JF, Chen QX, Tian HY, Gao X, Yu ML, Xu GJ, Zhao FK. Anal
Bioanal Chem 2008 Apr;390(7):1765-73
in a comparison of stains.
Desiderio procedure: R.R. Becklin, E.S. Umstor, D.M. Desiderio, poster WPF 216, 48th American Society for Mass Spectrometry Conference on Mass Spectrometry June 11-15 2000, Long Beach, CA
