How much protein have you loaded on the gel? Also, have you tried staining with coomassie or running a 1D gel to confirm that you have protein in the sample?

Thank all friends

In response to dear Andrew

Yes, I run a 1 D gel and then I stained it with CBB G-250. My bands are good enough. About the loaded concentration, I load 1 mg (at maximum concentration) on each IPG.

I think that Silver staining at the final step needs another reagent that prevent the loss of spot olor.

What do you think about that?

I hope that other researchers take part in this conversation to resolve the problem.

Cheers

First off, I'd say that seems a lot of protein for silver staining. Have you tried silver staining a 1D gel?

If you have coomassie G-250 I would try staining a 2D gel with that to make sure that your 2D methodology is ok. If you get spots (which you should loading 1mg) then it is obviously something with the silver staining protocol. If you don't then there is something wrong with your 1st or 2nd dimension running. Our protocol for G250 staining of 2D gels is below:

Fix the gel for at least 1 hour in methanol:acetic acid:water (5:4:1)

Stain the gel 24-72 hours in G250 solution (100ml water, 100ml orthophosphoric acid, 100g ammonium sulphate, 1g G250 and make up to 800ml stir then add 200ml methanol)

destain in water

Hope this helps

Dear my friend, Andrew

For checking 2DE, I stained my 2 DE gel first with CBB and after observing good spot, I put the gel in destaining solution for 72 hrs and then run silver staining. In this protocole, I found good spot but if I run 2DE and silver staining together, my results are not good enough.

Do you have other intresting ideas?

Hi, if you want more sensitivity that CBB. and your silver staining is not working good, you can maybe try with the modificated protocolo of coomasie.

Search this paper:

Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis.

Grettins

Low extraction and staining efficiency must be two of the factors that should be looked at closely