Hi,
Did anyone visualize fluorescent bands for fluorescent tagged proteins on a blue native gel? Do you destain the gel after the run?
If anyone has a working protocol I would appreciate it.
Thanks in advance,
Fawzi
I haven't tried it, but I think it will be difficult because of (1) background fluorescence from the dye and possibly also (2) quenching of fluorescence by the dye and (3) absorption of the excitation and emitted light by the dye.
You might be better off using native PAGE without the dye.
Thanks Adam, what about de-staining the gel, does it help? I'm avoiding doing a clear native because the proteins I'm studying are basic proteins. I'll be using Typhoon 9400 for imaging.
I don't think you will be able to remove the stain from the protein without using solvents that will also denature the protein. If it is OK to denature the protein (OK for covalent fluorophores, not OK for GFP-like proteins), then thorough destaining may work. Since destain is acidic, you will have to neutralize the pH to recover the fluorescence of some organic fluorophores, such as fluorescein.
Hi Fawzi,
I have done this with GFP tagged proteins and it worked after standard destaining of the gels, not the surfactant. If you are using precast gels/reagents, eg: from thermo, just follow the directions.
I also remember, although we had very highly over expressed proteins, the bands were somewhat visible even before we destained the gel; although the ladder we were using was not.
Best of luck!
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