I am doing FP with a large protein (170 kDa) and a smaller dimerizing protein (45 kDa/90 kDa dimer) with the same RNA oligo. Both bind with different affinities, but the max FP signal is the same for both experiments at ~200 mP. As polarization is dependent on the size of the protein, I was expecting a difference in max signal between these but didn't observe one. Is there a maximum size cutoff that can be detected via FP? So, would a 90 kDa complex have the binding as 170 kDa? What's the lower limit on this?
The relationship between fluorescence polarization and protein molecular weight depends on the fluorescence lifetime of the fluorophore. You can see a graph of this relationship here:
https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/technical-notes-and-product-highlights/fluorescence-polarization-fp.html#:~:text=Fluorescence%20polarization%20increases%20as%20molecular,the%20dye%20(%CF%84)%20increases.
Typical organic fluorophores have lifetimes of a few ns, which means that the polarization change is going to be the same for binding to a small protein or a large one, as you observed, all else being equal.
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