Figure 6. Evaluation of thrombin activity using “split luciferin approach”.

(a) Bioluminescent signal in function of the time from GGR-(D-Cys) peptide incubated with thrombin protease at different concentrations (5, 10, and 20 U) over 3 h at 37°C before addition of NH₂-CBT and additional incubation for another 1 h at rt. Signal acquisition was started right after addition of 5 μL of the pre-made luciferase buffer (see recipe) and continued for about 2 h using IVIS Spectrum (Perkin Elmer, USA). The blank represent the signal from luciferase buffer. (b) Total light output collected over the period of 2 h obtained by integrating the area under the curves in A and plotted in the form of bar-graph. Adapted with permission from Godinat, A., Park, H. M., Miller, S. C., Cheng, K., Hanahan, D., Sanman, L. E., Bogyo, M., Yu, A., Nikitin, G. N., Stahl, A., Dubikovskaya, E. A. 2013 A Biocompatible In Vivo Ligation Reaction and its Application for Non-Invasive Bioluminescent Imaging of Protease Activity in Living Mice, ACS Chem. Biol. 8, 987–999. Copyright 2014 American Chemical Society.