CPMSC Facility: Protocols and Forms
Mass Spectrometry Analysis Form
Proteomics Sample Submission Form
These procedures are compatible with mass spectrometry analysis of proteins. Quantities are for large (20 cm x 20 cm) gels.
||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 minutes
Wash twice in distilled water for 10 minutes
Sensitize 2 minutes with 0.5L of 100 mM sodium thiosulfate, 30 mM potassium ferricyanide
Wash in 0.5 L water for 10 minutes four times
Stain 20 minutes with 1 L 0.2% silver nitrate, 0.076% formalin
Wash in water 1 minute twice
Develop till dark enough with 1 L of 6% sodium carbonate, 0.05% formalin, 0.0004% sodium thiosulfate
Stop 5 minutes with 1 L of 50% MeOH/12% acetic acid
Store in 1% acetic acid/water
Water 5 minutes (3X)
Destain whole gel 15 minutes with 1:1 v/v 30 mM potassium ferricyanide and 100 mM sodium thiosulfate prepared fresh
Water 5 minutes (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