I would be grateful if someone would advise or give a link on a detailed fluorescence titration technique. I am interested in setting up an experiment on binding of a receptor to a fluorescein-labeled peptide ligand, choosing a control, etc.
Please look at the following below links which may help you in your analysis:
https://www.uslims.aucsolutions.com/fluorescence-binding.php
https://www.univie.ac.at/biologischechemie1/phw/Day2.pdf
Thanks
It would help to know some more details about your experiment.
- Are you going to use a purified receptor protein or a cell line expressing the receptor on the surface?
- What type of equipment for monitoring fluorescence do you have available?
- Do you want to use fluorescence intensity, fluorescence polarization, FRET, TR-FRET, or some other technique?
Adam B Shapiro We have a purified receptor and fluorescein labeled peptide as ligand. Device for simple flu-intensity measurement Fluoroscan 3. We placed peptide solution with defined concentration in cuvette and then made up titration with receptor solution. We calculated Kd but it is possible we observed the receptor-fluorescein binding instead of peptide-receptor binding. That is the question...
The way to determine if the signal was due to binding of fluorescein rather than peptide is to try to compete the fluorescein-peptide off the receptor using the unlabeled peptide.
Adam B Shapiro Yes, I think that's reasonable. Can you recommend any article or reference to this method?
Perhaps this paper of mine will help.
https://pubmed.ncbi.nlm.nih.gov/24820111/
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