I have been trying to do 2D-DIGE. I'm using Lumiprobe 3Dye™ 2D DIGE dyes for labeling, however after IEF and 2nd dimension, I found no spots on the gel. I tried to do the silver staining of the same gel and I saw spots on the gel. So for some reasons, labeling does not work. I used DTT in tissue solubilizing process and I am using regular fluorescent imager to scan the gel. has anyone had the same problem before?
Hi Mehdi,
CyDyes are extremely reactive once present in an oxygenated environment. Most CyDyes are shipped in dark black seal containers to prevent the "degradation of the dye". If your dyes are not being properly shipped, this could be one of the many reasons you are not getting proper labeling.
Additionally, are the dyes that you get already re-suspended in DMF or do you have to do that? When you re-suspend your dyes, are you using a fresh aliquot of DMF or old DMF? DMF will hydrolyze if left out on the bench for sometime in a standard mirocentrifuge tube.
Last, how long are you labeling the proteins for? Typically you can get away with a 15 minute reaction on ice, for minimal labeling purposes. How are you quenching the labeling reaction at the end?
I hope some of these questions will lead to a resolution for you.
Hi Malachi,
Yes dyes were shipped in dark sealed containers, but I re-suspend them in fresh aliquots of DMF. However, I've been storing them at -20C for about a year. I usually do 30 min labeling on ice and then quench the reaction using 10 mM Lysine. However, since 30 min labeling did not work, I tried even over night labeling at 4C that still did not work.
Hi,
Are you sure that there's no other (excess of) primary amines present in your reaction mixture, such as Tris?
Hi Mehdi,
I usually advise against re-suspending dyes in DMF and re-freezing them down for long term storage. DMF is highly reactive once oxygen is around and regular microcentrifuge tubes are not sealed tightly enough to keep the oxygen out. We've experienced issues with re-freezing re-suspended dyes and we suspect it was the DMF that oxidized even when frozen at -80.
Additionally, if your -20 freeze is a cycled, self-thawing freezer, then I would suspect this is the same problem you are having and that the re-suspended dyes went bad. I hope this isn't the case, but it certainly is plausible given the information you stated. If this is it, I would advise ordering new dyes, store dehydrated dyes at -80 and re-suspend as needed.
I hope this helps. Good luck with your experiments.
and....the presence of a reductant (hic DTT) must be avoided during labeling, as far as I remember....
There is not any primary amine in the solution, however, Thanks Malachi for your comments.
I think the reason for poor labeling is that the dyes has lost activity due to long storage time in reconstituted state. Amersham CyDye DIGE Fluor minimal dyes are stable for two months once reconstituted in DMF and stored in -20.
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