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CMS Resource: Protocols and Forms

Mass Spec Analysis Form

Proteomic Analysis Form

These procedures are compatible with mass spectrometry analysis of proteins. Quantities are for large (20 cm x 20 cm) gels.

Vorum Silver Stain Method

1. Fix 2-18 hrs. (50% Methanol/12% Acetic acid, 0.05% formalin), 1 liter/gel

2. Wash three times in 35% Ethanol/water for 20 min

3. Wash twice in distilled water for 10 minutes

4. Sensitize 2 min with 0.5L of 100 mM sodium thiosulfate, 30 mM potassium ferricyanide

5. Wash in 0.5 L water for 10 min. four times

6. Stain 20 min. with 1 L 0.2% silver nitrate, 0.076% formalin

7. Wash in water 1 min. twice

8. Develop till dark enough with 1 L of 6% sodium carbonate, 0.05% formalin, 0.0004% sodium thiosulfate

9. Stop 5 min. with 1 L of 50% Methanol/12% Acetic acid

10. Store in 1% Acetic acid/water

Desiderio Silver Destain

1. Water 5 min (3X)

2. Destain whole gel 15 min. with 1:1 v/v 30mM potassium ferricyanide and 100 mM sodium thiosulfate prepared fresh

3. Water 5 min. (10X)

4. Proceed to fixation procedure for staining or store at 1% Acetic acid/water

*Destain is useful if background darkens faster than the protein bands or if the gel is not stopped quickly enough and is generally too dark.

References:

1. 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.

2. 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.

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