Silver staining method

[dttom]

In silver staining, thiosulfate is used to sensitise the electrophoresis plate, for easier subsequent staining, but could anyone tell why and how thiosulfate can do this?

 

The protocol used is like this:

silver nitrate staining:

1) sensitised by thiosulfate;

2) immerse with silver nitrate;

3) apply mixture of formalin and potassium carbonate (carbonate what use? remove proton formed from oxidation of formalin?);

ammoniacal silver staining:

1) sensitised by thiosulfate or plate prepared with thiosulfate;

2) immerse with ammoniacal silver nitrate;

3) apply mixture of formalin and citric acid;

 

So, silver nitrate method and ammoniacal silver method differs; on application of formalin, in the latter case, ammonia seems to be pulled out from silver complex by adding citric acid, so I wonder if the property differences come from the process of application of silver solution, instead of in the process of reacting formalin. Would someone mind clarify this?

 

Thanks.

Edited March 17, 2010 by dttom

[CharonY]

I assume you mean silver staining as in silver staining of protein gels?

It is mainly used to reduce background staining. It is based on the complexation of silver that reduces unspecific precipitation.

[dttom]

Yes, that is silver staining for protein on gels (PAGE).

Silver is used to reduce background staining? Could you elaborate a little bit about this, I assume the silver ion (in silver nitrate) acts as an oxidising agent...

[CharonY]

The basic principle for theses stains is that silver is reduced to metallic silver which is then deposited on the analyte (e.g. proteins). Control of the kinetics of this reduction is crucial to avoid significant reduction in the matrix (i.e. PAGE).

By pretreatment of the PAGE the amount of free ions that may precipitate (especially during pH shift). This slows the reduction kinetics and allows a more specific staining (as the analytes are preferred nucleation sites).

Note that there are dozens of different flavors of silver stains out there some with significant modifications.

[dttom]

Sorry I could only barely grasp what you mean, could you see if I summarise it right?

 

"If the PAGE plate is pretreated with thiosulfate, upon formalin addition, thiosulfate and formalin form an equilibrium, to a degree preventing excess reaction between formalin and silver nitrate; however, in regions of protein spot, the protein would screen the thiosulfate nearby (thiosulfate content would be lower in those spots), together with proteins as a more suitable nucleating site, silver (metallic) would be mainly deposited on protein spots."

[CharonY]

Nope. The thiosulfate reacts with silver and forms a complex with it. Complexated silver is reduced slower than free.