I am measuring the binding of thrombin binding aptamer (TBA) to thrombin using fluorescence anisotropy. TBA is labelled with a fluorophore (5'-FAM) and was initially kept at a constant concentration of 30 nM while titrating in the protein at concentrations from 1 nM to 1 uM. When fit with the quadratic binding expression (so accounting for ligand depletion), I got a Kd
First, make sure your aptamer has not become degraded between the first and second experiment. Second, be sure to subtract the background fluorescence intensities (i.e. in the absence of the aptamer) before calculating anisotropies in the second experiment. At such a low fluorophore concentration, background is likely to be substantial.
Just to echo Adam's concerns, can you prepare trypsin (or chymotrypsin) in the same way you prepare thrombin and use that as background?
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