During binding studies the fluorescence measurement of proteins on adding ligand is not showing one pattern.it is firstly decreasing and then start increasing with increase in wavelegnth.
Binding studies usually use a fixed pair of excitation and emission wavelengths, then titrate either the protein or the ligand.
If you meant that the fluorescence first decreased then increased with increase in ligand concentration, however, then my suggestion is to perform the same titration again in the absence of the protein in order to see whether the increase in fluorescence is coming from fluorescence of the ligand.
Thanks Adam for reply .
So you mean simply in buffer I should see the fluorescence by increasing the ligand.
I tried my experiment many times using titration method taking low to high conc. of ligand but different pattern is coming.
Ligand is polyphenolic compound.
There are several potential artifacts that can complicate such experiments, especially with something like a polyphenolic compound.
The compound itself may be fluorescent.
The compound may act as a fluorescence quencher of the fluorophore.
The compound may significantly absorb the excitation and/or emission light. This is called the inner filter effect.
These effects are not mutually exclusive. The compound can have any or all of these effects. If the situation is not too complex, it is often possible to measure and correct for the artifact.
yes i perform an experiment and i found that my ligand fluorescence intensity is increasing without protein.
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