I am trying to study the interaction of my protein with a small molecule ligand by trp fluorescence spectroscopy. My protein has molecular weight of 16 kDa and has 2 tryptophan residues. I am keeping protein concentration 2uM and gradually increasing the ligand concentration (0-10uM). I am setting up separate binding reactions for each concentration of ligand. Excitation wavelength is 295 nm and emission spectra recorded from 310 to 400 nm. Excitation and emission slits both are 10 nm. Scan speed is 100nm/sec. The buffer used in the study has the following composition: 25mM HEPES-NaOH (pH 7.4), 150 mM NaCl and 10% (v/v) glycerol. The ligand is soluble in DMSO and I am keeping a final 2% (v/v) DMSO in all the reactions. I wish to find the binding affinity and stoichiometry from this study.
The problem is that I am not getting any definite pattern in the emission spectra of the protein upon increasing the ligand concentration; I am getting fluorescence enhancement for some ligand concentrations, for others I am getting fluorescence quenching. Please suggest a solution to this problem. (The protein and ligand interacts, that has been checked by other methods).
At first check the uv-vis and fluorescence Spectra of the ligands. In those cases where intensity increases, it may be due to the addition of fluorescence intensity of ligands with protein.
Hello.
You need to take the absorption and fluorescence spectra of each concentration of only ligand and subtract them from the corresponding concentration of ligand-protein. If your protein and ligand both absorb at the same wavelengths, UV and fluorescence are not very good methods for the study. You need to correct the absorbance for the inner filter effect as well as the fluorescence intensity by easily available mathematical equations.
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