Silver stain protocols

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.)

Vorum

Desiderio Silver destain to be used when restaining desired.

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

 

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

 

 

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