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In order to analyse the performance of our CT52 fused NanoBiT fragments, we measured the luminescence they produce at varying concentrations from 10nM to 10mM. We extracted the initial luminescence of the assays by fitting the light activities measured in time to the following formula:
In this equation y is the measured light output in absolute light units, t is the time in seconds, a is the initial light output, b is a multiplication factor that determines the range of the fit, and k is the decay rate of the substrate.
These initial outputs where then fitted to a one site specific binding formula to determine the maximal luminescence and the dissociation constant of our CT52-NanoBiT fragments.
After global optimization using metropolis Monte Carlo and Latin hypercube sampling the best fit resulted in LUmax = 3.5* 105 ±1.5 a.u. and KD = 996±163 μM (see figure 1), standard deviations are calculated with the Jacobian.