Some works correct with a factor which take into account the absorption data of the ligand and others works use the absorption data of the protein-ligand complexes. Which would be the correct form? We are performing measurements with constant protein concentration, i.e. vary the ligand concentration.
Please give more detail about the experiment. Is the protein or the ligand fluorescent? If the protein is fluorescent and the ligand is not but absorbs light, you can measure the inner filter effect by using a different fluorophore that the ligand doesn't bind to instead of the protein to measure the decrease in fluorescence caused by inner filter effect. For example, if you are measuring protein tryptophan fluorescence, use N-acetyltryptophanamide and the same excitation and emission wavelengths. At each ligand concentration, calculate a correction factor from the ratio of expected (no ligand) to actual fluorescence. In the experiment with the protein, multiply the fluorescent measurement at each ligand concentration by the corresponding correction factor.
Please give more detail about the experiment. Is the protein or the ligand fluorescent? If the protein is fluorescent and the ligand is not but absorbs light, you can measure the inner filter effect by using a different fluorophore that the ligand doesn't bind to instead of the protein to measure the decrease in fluorescence caused by inner filter effect. For example, if you are measuring protein tryptophan fluorescence, use N-acetyltryptophanamide and the same excitation and emission wavelengths. At each ligand concentration, calculate a correction factor from the ratio of expected (no ligand) to actual fluorescence. In the experiment with the protein, multiply the fluorescent measurement at each ligand concentration by the corresponding correction factor.
If it is possile to work in diluted solutions it is often not important to consider the inner filter effect. In most cases fluorescence signals are orders of magnitudes higher than absorbance.
The magnitudes of absorbance and fluorescence signals can not be compared. An absorbance value is the log of the ratio of incident light to detected light. Fluorescence intensity has arbitrary units: the scale has no physical meaning but depends on the instrument and its settings.
The concentration of a substance that produces an inner filter effect (IFE) in a fluorescence measurement depends on its absorbances at the excitation and emission wavelengths. If those absorbances are negligible, then no correction for IFE is needed. Generally speaking, absorber concentrations in the nanomolar range will have negligible IFE. Once you get into the micromolar range, it is more likely that there will be a significant IFE if the substance has absorbance at the excitation and/or emission wavelengths.
In addition to reduced fluorescence due to IFE, it is also possible to get static and/or dynamic quenching effects, which have nothing to do with absorbance, and resonance energy transfer. Moreover, the added substance could itself be fluorescent.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence