Silver staining of polyacrylamide gels

Reagents

• Fixation solution (50 : 5 : 45 v/v/v methanol : acetic acid : water)
• Sensitising solution (0.02 % sodium thiosulfate)
• 0.1 % AgNO₃
• Developing solution (0.04 % formaldehyde in 2 % sodium carbonate)
• 1 % acetic acid

Method

1. After the gel has been run, fix the protein by incubating the gel slab in fixation solution for 20 - 30 minutes.
2. Rinse the gel slab with water (2 changes, two minutes per change) and then leave it further in water for one hour on a shaking platform.^a
3. Sensitise the gel with sensitising solution for 1 - 2 minutes. Discard solution and quickly rinse the gel slab with two changes of water (10 seconds each).^b
4. Incubate the gel in chilled 0.1 % AgNO₃ for 30 minutes at 4^o C (fridge).
5. Discard silver nitrate solution and quickly rinse the gel with two changes of water (30 seconds per each change).
6. Develop the gel with developing solution. Discard the developing solution as soon as it turns yellow and replace it with a fresh portion.
7. When a sufficient degree of staining has been obtained, quench staining by discarding the developing solution and replacement with 1 % acetic acid. Wash the gel with 1 % acetic acid several times and store in the same solution.^c

^a Extended washing time helps to eliminate yellowish background usually observed after long developing of the gel (see step 6 of this protocol)
^b Agitate gently to make sure that the gel slab is covered evenly.
^c Silver stained gels can be stored at 4^o C in 1 % acetic acid for months. In some cases the colour of the stained protein bands might slightly change in time. However these changes do not affect the results of mass spectrometric sequencing.

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