Hi Aron,

I've had limited experience binding Alexa-Fluor molecules to proteins, but in theory you should be able to get this to work as long as whichever cysteine your attacking is far enough away from the matured fluorophore of GFP. GFP already has a few cysteines within it, so I'm not sure where your added cysteine will be or how that might affect the process. You may need to adjust the pH for this reaction to get more or less binding of the Alexa-Fluor to the cysteine(s). Thankfully GFP is a pretty stable protein, but it should be obvious with the lack of fluorescence if the tagging is affecting the maturation of the fluorophore. You could do a quick imaging test on a gel imager using the Alex-Fluor wavelength to see if it is bound, and double check with a SDS gel to see if the size of you GFP is increasing.

Best of luck,

Max

I have labeled several proteins with Alexa-flour and this should be possible, provided the caveats that Max describes. If you are doing this to increase the fluorescent signal in some assay, people often do this using anti-GFP antibodies....

Hi Aron,

Theoretically it is possible to tag Alexa dyes on the protein that already labeled with GFP. You have to use the maleimide version of Alexa dyes and maintain the pH of the solution between 6.5-7.5. We usually performed the labeling experiment in PBS (pH-7.2). Though, I haven't performed the dual tagging a protein before but it should work in a similar fashion. Though, it will be tricky not to tag the cys of GFP.

I don't know for which purpose you need this kind of dual tagging but you should know that this GFP and Alexa 568 acts as a very good FRET pair and as they are in a same protein they might act like it. When you will excite the molecule at GFP's excitation wavelength you will surely observe the emission of Alexa 568.

Good Luck

Prasenjit

One more thing, as sulfhydryls are prone to oxidize into disulfides they must be reduced by a reducing agent before conjugation with an Alexa dyes.

You can follow this article- https://pubs.acs.org/doi/full/10.1021/acs.bioconjchem.6b00371?src=recsys

Good luck

Prasenjit