Could anyone provide references or protocols to measure the binding affinity by using fluorescent ligand with protein? Ligand shows fluorescence excitation/emission: 270/340 nm.
I think maybe you can try to use fluorescence anisotropy measurement to determine their binding affinity. I don’t know how big your fluorescent molecule is. Let’s say, the fluorescent molecule is very smaller than the protein. Before binding of protein, the anisotropy of fluorescent molecule should be around zero due to its fast rotation diffusion in solution. After binding of protein, the mass of the complex is much bigger than individual fluorescent molecule. Therefore, its rotation becomes much difficult, resulting in a big anisotropy. If you titrate the fluorescent molecule with the protein, you will get a serial of anisotropy. According to the equation in this article (J. Phys. Chem. B 2002, 106, 452-459.), the binding affinity can be obtained.
The ligand has excitation/emission very similar to tryptophan. If it is present in your protein sequence it may cause problems in data interpretation.
Thanking you Adam Pomorski ,Yin Li . Target protein doesn't have tryptophan , Can I measure Steady-state fluorescence quenching by adding protein to Fluorescent ligand and which equation is best fit to measure binding constant and molecularity .
Dear Yin Li ,
Thanks for your reply .
At these wavelengths ( excitation/emission: 270/340 nm ) target protein absorption and emission is silent . so can I use Steady-state fluorescence quenching and could you please provide the link or reference for binding affinity calculation javascript if possible .
this is one of my old papers where we used the Benesi-Hildebrand plot for determining the binding constant and free energy changes in case of such protein-probe complexations as well as many other studies related to protein-probe binding. This maybe of some help to you.
Good luck !
https://www.researchgate.net/publication/24044743_Interaction_of_human_serum_albumin_with_charge_transfer_probe_ethyl_ester_of_NN-dimethylamino_naphthyl_acrylic_acid_an_extrinsic_fluorescence_probe_for_studying_protein_micro-environment?ev=prf_pub
Article Interaction of human serum albumin with charge transfer prob...
Hi Rupashree Singh ji .
Thanking you for great help . I finished my experiments as per your Publication . We observed blue shift while titration of protein to ligand . The ligand binding site is hydrophobic pocket of the protein .Result is consistent with My NMR experiments. we got the extent of binding K ( Extent of binding ) from the plot 1/[I - I0] vs. 1/[protein] . Could you please instruct us to calculate the Kd ( dissociation constant ) and Delta G from K ( Extent of binding ) .
Thanking for your great help and publication .
Good to know that Srinivas. I don't remember estimating Kd anytime but I think it's just the reciprocal of K (binding constant). But some do that using Scatchard plot.
& deltaG can be estimated using -RTlnK.
Hope this answers your query.
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