Silver staining of proteins and nucleic acids in polyacrylamide gels
250 ml of each solution are used for one big gel. The solutions containing thiosulfate have to be freshly prepared in order to get sensitive and reproducible staining. All steps of the silver staining procedure are performed in plastic container on an orbital shaker at room temperature.
The image is unstable at room temperature, but the stained gels may be stored for few weeks at 4C in 50 MeOH or in 1 AcOH with only a minor loss of image quality. For detailed description see: Blum H., Beier H., Gross H.J., Electrophoresis 1987, 8: 93-99. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels.
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Steps |
Solutions |
Time of treatment |
|
1. Fix |
50 MeOH (125 ml), 12 AcOH (30 ml), H2O (95 ml) |
≥ 1 h, also over night |
|
2. Wash |
50 EtOH |
3 20 min |
|
3. Pretreat |
0.02 Na2S2O3 5H2O (0.05 g) |
1 min |
|
4. Rinse |
H2O |
3 20 sec |
|
5. Impregnate |
0.2 AgNO3 (0.5 g), 187.5 microl 37 HCOH |
20 min in dark! |
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6. Rinse |
H2O |
2 20 sec |
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7. Develop |
6 Na2CO3 (15 g), 125 microl 37 HCOH, Na2S2O3 5H2O (1 mg) |
≥ 10 min in dark! |
|
8. Wash |
H2O |
2 2 min |
|
9. Stop |
50 MeOH (125 ml), 12 AcOH (30 ml), H2O (95 ml) |
10 min |
|
10. Wash |
50 MeOH |
≥ 20 min |
MeOH – methanol, AcOH – acetic acid, EtOH – ethanol, HCOH – formaldehyde.
Important! Sensitivity is about 1-10 ng! Silver stains the proteins starting from the surface of the gel! The chemistry, involved in silver staining and silver removal, may modify the proteins: block the N-terminus!
2004 02 11 – Julius
Destaining of silver stained gels
This protocol is suitable for over-developed or with a high bacground gels. Silver stained proteins have also to be destained prior to enzymatic digestion.
Chemicals:
Na2S2O3 5H2O, Fw 248.2 g/mol
K3[Fe(CN)6], Fw 329.25 g/mol
Solutions:100 ml250 ml500 ml
Solution A: 10 mM sodium thiosulfate pentahydrate 248.2 mg 620.5 mg 1.24 g
Solution B: 30 mM potassium ferricyanide 987.75 mg 2.47 g 4.94 g
Mix both solutions in a ratio 1:1 just before the destaining!
Protocol:
1. Rinse the gel with distilled H2O.
2. Add destaining solution: 250 ml/gel.
3. Shake until the silver staining disappears (about 20 min).
4. Wash with distilled H2O (2 x 5 min).
5. Wash with STOP solution (50% Methanol, 12% AcOH) until the yellow colour of the gel almost disappears (or overnight at 4ºC; change STOP solution several times).
7. Start the silver staining from the washes in 50% Ethanol (Step 2).
The destaining mechanism is as follows: potassium ferricyanide reacts with silver and forms silver ferricyanide, and that reacts with thiosulphate to form water soluble complex.
For detailed description of the method see: Gharahdaghi F., Weinberg C.R., Meagher D.A., Imai B.S., Mische S.M. Electrophoresis, 1999, 20 (3): 601-605. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity.
2003 06 20 – Mageni & Julius