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Silver Staining Principle

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exec
India

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Topic Started by exec
on 12/9/2006 0:49 AM   
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we use non ammoniacal silver staining using reagents as Na2S2O3, AgNO3 + Formaldehyde, developer as Na2CO3 + formaldehyde , stop solution as citrate.

can someone please explain me the principle of using this reagent in silver staining or mechanism behind silver staining.


it will be of great help. thanx,


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Tony Rook
United States

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Posted By Tony Rook
on 1/9/2007 5:24 AM   
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exec:

The following link has a good summary of these historic silver staining protocols:

Two modifications to the silver nitrate staining technique of Merril et al, 1981
- Blum et al, 1987
- Heukeshoven et al, 1988

And the the silver diamin procedure of Oakely et al, 1980


http://www.weihenstephan.de/blm/deg/manual/manualwork2html02testp7.htm

Introduction:

Silver staining methods are about 10-100 times more sensitive than various Coomassie Blue staining techniques. Consequently, they are the method of choice when very low amounts of protein have to be detected on electrophoresis gels. A huge number of silver staining protocols have been published, based on the silver nitrate staining technique of Merril et al. (1981) and modifications (e.g. Blum et al, 1987, Heukeshoven et al. 1988) or the silver diamin procedure of Oakely et al. (1980).

It advisable not to pre-stain gels that will be subjected to autoradiography, as it will cause quenching (this is more a problem with silver staining than with Coomassie staining). Gels are soaked in an autoradiography reagent for 30 min at room temperature if using 35S or 14C labelled proteins, whereas gels containing 32P labelled proteins do not require such a treatment.

Electrophoretically separated polypeptides can be visualized by "general" stains like Coomassie Blue, silver, fluorescence or autoradiography, or by "specific" stains such as glycoprotein staining or immunochemical detection methods. Whereas the "general" protein stains are carried out in the electrophoresis gel directly, immunochemical detection methods are usually performed after electrophoretic transfer ("blotting") of the separated polypeptides from the electrophoresis gel onto an immobilizing membrane (Towbin et al. 1979; Kyhse-Andersen 1984).

Since the advent of highly sensitive microsequencing techniques, it is also possible to gain N-terminal or even internal amino acid sequence information of proteins separated by 2D-PAGE. Coomassie Blue stained spots ecxised from the electrophoresis gel or the blotting membrane with a scalpel or a razor blade can be subjected directly, or after cleavage, to Edman degradation using a gas phase amino acid sequence analyzer, amino acid composition analysis, or mass spectrometry. Usually, a single Coomassie stained spot yields sufficient protein (1-10 g) to obtain an N-terminal amino acid sequence. To elucidate internal sequences (which requires higher protein amounts), several identical spots from different 2-D gels have to be pooled. For mass spectro-metric analysis, lower amounts of protein (one silver stained spot) are often sufficient (Wilm et al. 1996).

Tony Rook



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