The ECs and QYs were determined as described6 (link),11 (link). The Soret band absorbance at 380 nm determines the protein concentration for the EC. The 380 nm absorbance is unchanged when covalently attached to smURFP or free in solution. To confirm no change, we denature an FP with a chromophore (BV or PCB) with 1 M urea. A Jasco V-770 UV/Visible Spectrophotometer measured absorbance. The Q band EC is determined by:
EC Max λ = Absorbance Max λ / (Concentration * Path Length)
The QYs were determined by comparison to Cy5, where QY = 25% in PBS. The absorbance of Cy5 and FP were matched using a Jasco UV/Visible Spectrophotometer. A Horiba Fluoromax-4CP Fluorometer measured the Cy5 and FP emission excited at the same wavelength. The FP emission was integrated and divided by the integral of the Cy5 emission and then multiplied by QY = 25%.
A Digital Frequency Domain system (ChronosDFD, ISS) measured the fluorescence lifetime of smURFP+BV in PBS. Chromophores were excited with a 518 nm laser diode (ISS). Fluorescence was detected after a 561LP filter. Rose Bengal in ethanol was the reference with τ = 0.785 ns66 (link) to obtain the instrument response function. The fluorescence decay was fit to a single exponential with χ2 = 0.98.
EC Max λ = Absorbance Max λ / (Concentration * Path Length)
The QYs were determined by comparison to Cy5, where QY = 25% in PBS. The absorbance of Cy5 and FP were matched using a Jasco UV/Visible Spectrophotometer. A Horiba Fluoromax-4CP Fluorometer measured the Cy5 and FP emission excited at the same wavelength. The FP emission was integrated and divided by the integral of the Cy5 emission and then multiplied by QY = 25%.
A Digital Frequency Domain system (ChronosDFD, ISS) measured the fluorescence lifetime of smURFP+BV in PBS. Chromophores were excited with a 518 nm laser diode (ISS). Fluorescence was detected after a 561LP filter. Rose Bengal in ethanol was the reference with τ = 0.785 ns66 (link) to obtain the instrument response function. The fluorescence decay was fit to a single exponential with χ2 = 0.98.
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