pH sensing with fluorophores - (Oct/10/2011 )

Hey guys, I hope this is the right forum. I figured some of you cell biologists out there might have some experiences with fluorophores and the like.

I do not know if this is the best approach, but what I would like to do is perform an in vitro assay with a protein complex + substrate and detect a deprotonation event during catalysis. I do not want to conjugate any fluorescent groups onto the protein because I do not know if this would effect the chemistry. I decided that it may be possible to have a pH sensitive fluorophore in solution that can detect such a local change H+ concentration.

I have no clue about the feasibility of this kind of approach or if there is a better/standard way to detect local changes in pH or deprotonations.

The other problem is sensitivity and choosing the right fluorophore for the job. I have no idea where to even begin and I don't know the nuances that separate the different kinds of pH-sensitive fluorophores that are available. Additionally, I see some that are used for endocytosis for example yet still detect acidic pH changes. Could this still work for my purposes? There are also a number of papers listing synthesis of newer fluorophores but I don't know if these are tested and well used.

***Invitrogen lists some acidic pH fluorophores here.
***Invitrogen table that lists various fluorophores here.
***Sigma has some as well found here.

So I just don't even know where to begin. Any help at all would greatly be appreciated! Thank you!

Have a look at the Molecular probes handbook (on the invitrogen website), it typically has some good information on the sorts of experiments you can do with particular types of probes, and the associated chemistries.I

I suspect that even if you can find a probe that would potentially tell you of a localised pH change, that they would not be sensitive enough to tell you of a single deprotonation event, unless it is done in bulk.

You might be better off with a luminescence based system - pH change activates a luciferase or something?