I have done Coomassie Brilliant Blue (CBB) staining for protein detection then destained the gel for removal of excess dye not bound to proteins.
Now I want to remove the dye bound to proteins to go for silver staining with same gel. But in the destaining solution the dye bound to proteins will not remove completely. Could you suggest any other opinions?
The combination of both staining methods doesn't work well. Either CBB or silver. You will lose a lot of sensitivity. If you would like to compare different staining methods do this on western blots. So you can run your protein mix in a single slot, transfer it (best by electro-transfer in tanks). Cut the NC-membran into several trips and stain them differently. Ponceau-S, Silver, Gold, for carbohydrates, lipids, protein, by immune reaction ...
try microwave quick destaining method...at lowest temp i.e., 160deg C, put the gel (immersed in destaining solution) for 30 seconds in the microwave. 2-3 times should be sufficient.
The combination of both staining methods doesn't work well. Either CBB or silver. You will lose a lot of sensitivity. If you would like to compare different staining methods do this on western blots. So you can run your protein mix in a single slot, transfer it (best by electro-transfer in tanks). Cut the NC-membran into several trips and stain them differently. Ponceau-S, Silver, Gold, for carbohydrates, lipids, protein, by immune reaction ...
Mr. Santosh Kodgire, Can you tell me the proportion of destaining solution (Methanol:Acetic Acid:Distilled Water) Destaining solution
a. Methanol : 80 ml
b. Acetic acid : 20 ml
c. Distilled water : 100 ml
Use this proportion and if you want remove excess amount dye, you can increase the methanol and you can achieve the destaining of protein.
If you are just giving it a try, just stain with silver. The brown signal of the silver will cover the blue from the commasie. But as the ones above told you: this gel will not be publication quality and you might have to do it again.
For the next time, I will recommend you to do a negative staining (Zink staining is beautiful!) and if you still not see it, de-stain the gel and do silver. Here the sensitivity should not change much since the proteins were actually not stained with the zink.
Good luck
You could try to destain several times to remove the excess of comassie... when you see the gel is more clear, then you should try the silver... but it will not work 100% perfect...
Dear Mr. Santosh I also agree with the comments of Dr. Stephan Kiessig. However you could also do the things as per Mr. Murugan Rajenderan suggested. It will definitely destain the bound stain.
Thank u very much for your kind suggestions i had tried 40:10:50 (methanol:aectic acid:D/w) for destaining of gel still some cbb remain with protein but with several times destaining bound cbb loses protein and more transparent gel will produce then i go for silver staining but not that much resolution i got
next time i will try for the negative staining (Zink staining suggested by Luisa F. Jimenez )
i will also try the microwave destainig method kindly suggested by Siddhi Gupta for next time what will be the temperature suggested by you?
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