Characterization of Protein by Silver Staining

Preparation:

1. Kit components are stable for 6 months at 2-8^o
2. Prepare each reagent immediately prior to use.
3. Use nanopure water (18 megohm/cm) to prepare reagents.

Solution	Component	Volume (mL)
Fixing Solution	Nanopure water	21.4
	Methanol	23.8
	Acetic Acid	4.8
Sensitizer (contains glutaraldehyde):	Nanopure water	25.0
	Methanol	23.8
	Sensitizer	1.2
Staining Solution Stainer A (contains silver nitrate) Stainer B (contains ammonium hydroxide & sodium hydroxide)	Stainer A	2.5
	Stainer B	2.5
	Ultrapure water	45.0
Developer (contains formaldehyde & citric acid)	Nanopure water	47.5
	Developer	2.5
Stopper (contains citric acid)	Stopper	2.5

Note: Volumes above are for staining one gel. Multiply volume by number of gels for staining additional gels.

 

Procedure:

Note: The following incubation times are for 1-mm gels. For 1.5-mm gels, incubate each step for twice as long.
Note: Gels may be incubated in the second sensitizing solution overnight.

1. Fix the gel in Fixing Solution for 10 minutes.
2. Decant the Fixing Solution and incubate the gel in Sensitizing Solution for 30 minutes.
3. Decant the Sensitizing Solution and incubate the gel in another 50 mL of fresh Sensitizing Solution for another 30 minutes.
4. Decant the Sensitizing Solution and rinse the gel twice with nanopure water: 5 minutes (each rinse) for Tricine gels and reduced samples; 10 minutes (each rinse) for NuPage Bis-Tris gels.
5. Incubate the gel in Staining Solution for 15 minutes.
6. Decant the Staining Solution and rinse the gel twice with nanopure water for 5 minutes
7. Incubate the gel in the Developing Solution until the desired staining intensity is achieved, typically 30 seconds – 5 minutes. Add the Stopping Solution to stop development and incubate for 10 minutes.
8. Decant the Stopping Solution and rinse the gel three times with nanopure water for 10 minutes (each rinse).