Hi,

Are you looking to digest the PAGE-separated (and cut out) protein band and analyze the resulting peptide mixture? If so, you can first destain the gel pieces containing the silver-stained protein band using a solution of 30mM potassium ferricyanide and 100mM sodium thiosulphate.

Subsequently, you can use one of the many publicly available in-gel digestion protocols. In some cases, a speedvac is used to completely dry the gel pieces eventually, but this is not absolutely required. If you don't have a speedvac, you can just leave the gel pieces to dry for a while in a fume hood before adding the trypsin solution that will digest the protein in gel in the final step.

Hope this helps, good luck! 

An alternative to SpeedVac to dehydrate gel pieces is to wash them with pure acetonitrile. 

Note that not all silver staining methods are compatible with in-gel digestion as the more sensitive silver staining protocols cross-link the proteins to the gel.

You may use 1% hydrogen peroxide for destaining PAGE gel (silver stained). An example of the protocol can be found on page 9 at : http://www.bio-rad.com/webroot/web/pdf/lsr/literature/LIT-442.pdf

Dear Vandhana

Using silver stain usually requires a step where formaldehyde is added to attain the staining effect. This also cross links the protein with itself and other proteins in the gel band which is very seldom a single protein.

This can result in strange sequences being identified and reduce the number of positive peptide hits for the peptides in a digested protein.

It would be better to stain part of a gel with silver (you can cut it to size) and identify which bands you want to put through your MS and use the equivalent regions of the unstained part of the gel. Using a Coomassi counter stain will help identify the position of the bands which can also be Coomassie stained.

Use of fluorescent dyes can also provide the sensitivity of silver staining without the crosslinking problem.

The requirement of a speedvac only makes the process faster but be careful of just leaving the gels to dry in a fume hood or on the bench as contamination is a problem. You can use a vacuum desiccator or just a plain desiccator with a suitable sorbent and although it takes longer it works fine.

Best of luck

Thanks a lot for all your valuable suggestions..It is indeed of great help.Even I have read literature where they say silver staining hinders sequencing.But I cant see my desired band in Coomassie. So am bound to do silver staining and proceed