Hi all,
I'm interested on monitoring ATP binding to a protein of interest using fluorescent ATP analogs, such as TNP or MANT-ATP. Do you have any experience with them? Which one is the best option for this purpose? I've read that TNP-ATP only emits fluorescence upon binding to the protein, but I don't know if there is any other difference that would make TNP a better choice than MANT.
Thank you all in advance.
Ana
TNP has longer wavelength excitation and emission than MANT, which can be an advantage for some purposes. Both are modified on the ribose moiety, so in that sense they are similar. If the protein interacts with the ribose sugar, then both probes will alter that interaction. You should also consider trying probes that alter other parts of the ATP molecule. Remember that a fluorescent analog of ATP is not necessarily going to behave the same as ATP itself.
Etheno-ATP is a nucleotide with the fluorophore at the base:
https://www.jenabioscience.com/nucleotides-nucleosides/nucleotides-by-structure/fluorescent-nucleotides/adenosines-intrinsically-fluorescent/nu-1103-etheno-atp
TNP-ATP and formycin-nucleotides are classical monitors of ATP site in ATPases...FITC is known to be a good monitor of ATP binding site in ATPases through binding at some lysin residues nearby the catalytic triad...Is this your protein of interest capable to hydrolyze ATP? Is it inhibitable by vanadate or fluoride?
Thank you all
Carlos Frederico Leite Fontes We hypothesize that our protein could bind or hydrolyze ATP, but we actually don't know it for sure, that's what we want to confirm or discard by doing this type of analysis with ATP analogs. We are also thinking in using an ATP detection kit, could you recommend any?
Thank you again for your advice!
Luciferin-luciferase kit can be used to measure ATP hydrolysis but it is expensive... Some assays using phospho-molibdate complexes can also be employed...The protocol described by Fiske and SubbaRow, 1925 measure the Pi liberated from ATP hidrolysis... You should use vanadate as a control inhibitor, just in case to charachterize if it is an ATPase! One alternative way is to use 32Pi-ATP and measure 32Pi liberated from rediolabeled nucleotides...
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