Author Topic: Problems with silver staining  (Read 14711 times)

anna

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Problems with silver staining
« on: July 13, 2005, 05:00:00 PM »
I stained my gel with silver nitrate solution 0.1% + 0.076% formalin. After developing it took really long to see poorly stained spots (I did in parallel a Coomassie staining on onother identical gel and spots are there!). What I notice is a precipitating brownish powder on the gel and on the box. I prepared yesterday my silver nitrate solution, but now it is all precipitated on walls and bottom of the bottle even if I kept it in the dark.
Any explenation for the poor staining?
Thanks
Anna

Ambort

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Re: Problems with silver staining (anna)
« Reply #1 on: July 13, 2005, 05:00:00 PM »
Dear Anna,
How should I interprete your indications of silver staining? You use a solution that contains silver nitrate and formalin? Do you do your silver stain in one step? Do understand how silver staining works? It is a multistep procedure that consists of several steps and reagent solutions. Firstly, silver nitrate solution should be made up freshly, just prior to use without addition of formalin during staining. The principle of silver staining comes form photo technology. The Ag+ of silver nitrate binds to aminoacid residues of proteins and acrylamide matrix. During development the formaldehyde acts as reducing agent to reduce the Ag+ to elemental silver that appears black. This "crystallisation" process more likely starts up at higher Ag+ concentrations, namely those next to proteins compared to the concentrates in the background. The formaldehyde is oxidized to formic acid. The sodium thiosulfate present in sensitizing steps and some silver nitrate solutions keep the Ag+ in solution and diminishes unspecific staining of background. Anyway a neverending development would result in fully black gel.

A good protocol is Silver staining according to Swain and Ross. Electrophoresis. 1995 Jun;16(6):948-51.
An older widely distributed Silver staining protocol is according to Blum et al. Electrophoresis. 1987; 8:93-99.

greetings

Ambort


Reiner Westermeier

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Re: Problems with silver staining (Ambort)
« Reply #2 on: July 14, 2005, 05:00:00 PM »
Are you using plastic trays? You should only use glass or stainless steel trays, not plastic trays.
I know, of course, that there is some plastic material, which could be used for silver staining. But you need to find it. Most of the plastic material contains softeners (sulphur-compunds) which reduce silvernitrate to metallic silver.

anna

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(No subject)
« Reply #3 on: July 14, 2005, 05:00:00 PM »
Yes, I am using plastic trays from Nalgene constructed of PCTG [poly(1,4-cyclohexylene dimethylene terephtalate)glycol]. They are designed forstaining electrophoresis gels, but in effect it is not specified for silver staining!

Sjouke Hoving

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Re: Problems with silver staining (anna)
« Reply #4 on: July 20, 2005, 05:00:00 PM »
Check your reagents, probably too old (polymerized) formaldehyde.

Sjouke


JNK

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Re: Problems with silver staining (sjouke)
« Reply #5 on: July 24, 2005, 05:00:00 PM »
good pointg, I also had problem, when using too old formaldehyde