When I titrated fluorescence probe by Protein A alone or by Protein B alone, it do not should any binding signals, mP do not change. However, when I titrated the same fluorescence probe by Protein A and Protein B mixture (1/1 ratio), it always give a significant binding signals, mP has >100 change, regardless of what the proteins it is.
Please look at the following below links which may help you in your analysis:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277431/pdf/nihms334221.pdf
http://calvino.polito.it/~gasparini/fp_introduction1.pdf
https://research.fhcrc.org/content/dam/stripe/hahn/methods/biochem/beacon_fluorescence_guide.pdf
Thanks
Check to see that the apparent fluorescence polarization change is caused by binding of the probe to the protein mixture, and not to light scattering by the protein mixture itself. This is done simply by leaving out the fluorescent probe.
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