There is an alternative method that does not rely on hydrphobic interaction of dye with the protein interior: it relies instead on reaction between fluorogenic dye and interior cysteine residues of the protein. When protein melts these Cys residues become exposed, react with the dye and generate fluorescence. It works very well, with two caveats: 1) the really good yellow dye is not commercial, last time I checked (there is a masked blue dye, but it's not as good and it requires excitation in UV) and 2) you need 'unusual' excitation and emission wavelengths.
I have not checked recently, maybe someone developed a nice green or red emitter, and then this is an ideal method for membrane proteins and anything else that requires detergents... I believe the method was first reported by one of the GPCR-structure teams, who used the blue version -- I tried it a long time ago with the yellow version made for me by a friendly chemist. There was a chinese paper describing the dye synthesis (it was a Michael-reactive double bond that masked the fluorophore). Cheers, Artem - Cosmic Cats approve of this message On Sat, Apr 12, 2014 at 3:38 AM, Theresa Hsu <theresah...@live.com> wrote: > Dear all > > Does anyone has experience with Thermofluor assay to find the substrate > transported/binding by a membrane protein? My protein does not have any > similar structures and the substrate suggested by sequence analysis is not > being transported in proteoliposome. I know ITC is good but I am looking > for a more high-throughput way. > > Thank you. >
