Fluorescence - (Dec/02/2005 )
I am novice in protein fluorescence methods. I wonder why people does label proteins with a fluorescent probe to study changes in fluorescence intensity. Is not enough to study changes in intrinsic fluorescence?
Thanks a lot
i think people use fluorescent tags depending on the situation; for eg if you are trying to study protein interactions then tagging them is far more efficient than intrinsic fluorescence, but unfolding studies can be simply done using intrinsic fl. also, it depends on the position of the Trp, tyr residues in the protein you are studying. if you find that they don't offer any noticeable changes with the conditions of the experiments then you need to think about adding an external fluor like ANS or acrylodan or something to determine what the structure of the protein is, how hydrophobic patches are exposed etc. i know this answer isn't very comprehensive but the main advantage of fluorescence is it's incredible sensitivity to environment. when you change the environment (pH, hydrophobicity) there should be some measurable change in the fluorescence signal. if you find that you can't see one with intrinsic fluorescence then you should think about adding an external fluor, either post-translationally like ANS, or Texas red or if you can, make a fusion protein with a GFP variant. it's upto you and how you want to plan your experiments.
but if you get relevant changes just using intrinsic fluors then go ahead and use it. it's just as good.
Ok Soraya, thanks a lot. I would want to use fluorescence to study proteins interaction. Let me a last question: When using intrinsic fluorescence, if both interacting proteins have Trp residues the observed fluorescent changes upon interaction would be caused by both proteins, isn'it? So what about background?
The wavelengths of excitation and emission are different for different fluors requiring different lasers for excitation and filters for detecting the emission. You need to check your fluorescence microscope, plate reader or flow cytometry has the necessary set up for your fluor.
Most people use fluorescence microscopes and flow cytometers with FITC/GFP, some of the GFP variants (CFP/YFP) need additional lasers/filters.
All the best,
yes both proteins would fluoresce if they both have trp residues. Never done what you are doing so I'm not very sure but trying to distinguish between them may be a problem unless the environment of the Trp residues within each protein is vastly different. I think if you are interested in studying the conformational changes in each protein you could try chemical modification of one protein to quench it's Trp fluorescence using N bromosuccinimide or some other Trp modifier, then use the modified protein(which no longer fluoresces) in interaction studies with the other protein and observe the changes in the unmodified proteins fluorescence.