Hi Punith,

Classical silver staining includes the sensitizer "Glutaraldehyde"  and the original (classical) silver staining can usually detect down to about 1 ng of protein.

Mass spectrometry compatible silver staining utilizes "Sodium thiosulphate" as sensitising agent, before the staining step.  The mass spectrometry compatible silver staining is less sensitive than the original, classical method and can detect down to about 6 ng or protein.

I am not sure what the "rapid silver staining" is.  Most vendors sell kits and include eg., short washing steps or even microwave utility to shorten the staining.  Is this the "rapid" protocol?

When silver staining, it is important to use glassware that is free of protein.  To remove any residual protein, just rinse the glassware with ~80% glacial acetic acid, followed by water, followed by methanol, followed by water.  This will remove any protein on the surface of the glassware.  Remember that autoclaving kills germs, does not remove proteins from your glassware.

The second very important factor is to use very high quality of water.  MilliQ filtered water should be fine.

Here is a protocol that describes how to "bleach" silver stain:

Good luck!

Hediye

Dear Hediye,

Thank you for the answer. Rapid silver staining is perhaps the same what you are described above which uses sodium thio-sulphate for sensitizing (please refer to the attachment) which works best with us. During fixation of proteins, is there any advantage (or disadvantage) of using ethanol : acetic acid over methanol : acetic acid, also the time required for sensitizing. In some protocols they sensitize for 60 seconds followed by washing, in few they sensitize for 30 minutes (unusually longer). Kindly enlighten me on this issue.

Dear Punith,

I have always used ethanol:acetic acid.  Just have to make sure that it does not contain denaturants such as aldehydes.  Since this product is expensive and may have special storage regulations, I think methanol option should be explored.

I also used longer sensitizing time ( 30 minutes or longer).  I looked at the attached; make sure that when you are developing, you discard the first solution you add.  You will notice the cloudy appearance of the developing solution.  Discard the first solution that your gel is exposed to since the chemical is reacting to elements on the surface of the gel.

Best,

Hediye.

Dear Hediye,

I'll give a try with your suggestions. Mean while if possible,please share your protocol with me so that I can be benefited in my research.

Thank You.

Hi Punith,

Here is the protocol we have been using in the lab.  It also includes the "bleaching steps". 

All the best,

Hediye

Dear Hediye,

Thank you for sharing your lab protocol. It will be of great help for me.

Punith

Always used Shevchenko or a modification but that was years ago

http://www.ncbi.nlm.nih.gov/pubmed/8779443

Hi,

I am not sure about your research budget, but if it allows, I would recommend ProteoSilver™ Plus Silver Stain Kit from Sigma. It has option of destaining bands. I recently used it and our MS machine didn't complain