Figure 5.

Performing baseline correction of emission-ratio time courses

Due to the low fluorescence intensities associated with tagged proteins expressed at endogenous levels, photobleaching of the reconstituted GFP may significantly contribute to FluoSTEP emission ratio measurements in the form of a drifting baseline. One method to correct for baseline drift is to perform a linear fit of the baseline (i.e., time points preceding drug addition), after which corrected emission ratios (R_corrected,t) can be calculated by subtracting the fitted slope (slope_fit) from the original emission ratio (R_raw,t) value at each time point t. Data shown represent the mean±SEM.