Human Anti-fluorogen (TO1-2p) Recombinant Antibody (Clone AM2.2) (CAT#: PABS-0035)

Figure 1 Concentration Response Curve for Probe ML342 (CID 2953239) in AM2.2-β2AR/TO1-2p (fluorogen) assay

Wu, Y., Tapia, P. H., Gouveia, K., Sklar, L. A., Fisher, G. W., Waggoner, A. S., ... & Stauffer, S. R. (2013). Inhibitors of FAP-fluorogen interaction as a multiplex assay tool compound for receptor internalization assays. Probe Reports from the NIH Molecular Libraries Program [Internet].

Figure 2 Confocal microscopy comparison of AM2.2-β2AR- (A-C) and β2AR-GFP- (D-F) expressing cells treated with ML342.

Cells were maintained in the resting state (A, D), treated with 20 µM Isoproterenol (ISO) for 90 min at 37 °C (B, E), or treated with 10 µM ML342 for 90 min at 37 °C (C, F). For AM2.2-β2AR cells, 1 µM TO1-2p was added to the cells before the addition of ISO or ML342.

Wu, Y., Stauffer, S. R., Stanfield, R. L., Tapia, P. H., Ursu, O., Fisher, G. W., ... & Sklar, L. A. (2016). Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair. Journal of Biomolecular Screening, 21(1), 74-87.

Figure 3 Equilibrium binding of TO1-2p and ML342 to cell surface-accessible AM2.2 tag at 4 °C.

TO1-2p binds to surface AM2.2 tag with an affinity of ~2.3 nM. Median channel fluorescence (MCF) from AM2.2-expressing cells was measured by flow cytometry.

Wu, Y., Stauffer, S. R., Stanfield, R. L., Tapia, P. H., Ursu, O., Fisher, G. W., ... & Sklar, L. A. (2016). Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair. Journal of Biomolecular Screening, 21(1), 74-87.

Figure 4 Increasing concentrations of ML342 mixed with 2.5 nM, 25 nM, and 250 nM TO1-2p were added to AM2.2- β2AR cells on ice, and the affinity of ML342 to AM2.2 from the set of dose-response data was fitted to 155 ± 43 nM.

Wu, Y., Stauffer, S. R., Stanfield, R. L., Tapia, P. H., Ursu, O., Fisher, G. W., ... & Sklar, L. A. (2016). Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair. Journal of Biomolecular Screening, 21(1), 74-87.

Figure 5 Real-time association of TO1-2p binding to AM2.2.

AM2.2 cells resting on ice for 60 min before the experiment. After measuring the baseline fluorescence for 15 s, TO1-2p was added to the cells at time = 0 s. The final concentrations of TO1-2p are 2.5 nM (a), 5 nM (b), 7.5 nM (c), 30 nM (d), and 250 nM (e). The fitted association and dissociation rate constants using the built-in Prism model for this data set were 1.54 × 10⁶ M^-1 s^-1 and 0.0027 s^-1, respectively, and the R2 value for the five sets of data is 0.97, 0.95, 0.98, 0.82, and 0.67.

Wu, Y., Stauffer, S. R., Stanfield, R. L., Tapia, P. H., Ursu, O., Fisher, G. W., ... & Sklar, L. A. (2016). Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair. Journal of Biomolecular Screening, 21(1), 74-87.

Figure 6 Real-time association of TO1-2p to AM2.2-b2AR cells accompanied by ML342-induced dissociation.

Association was measured for three concentrations of TO1-2p (2.5 nM in purple, 7.5 nM in blue, and 25 nM in green) followed by addition of ML342 at 300 and 600 s. The on-and-off rate constants were fitted to 1.46 × 10⁶ M^-1 s^-1 , and 0.0041 ± 0.00031 s^-1, respectively.

Wu, Y., Stauffer, S. R., Stanfield, R. L., Tapia, P. H., Ursu, O., Fisher, G. W., ... & Sklar, L. A. (2016). Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair. Journal of Biomolecular Screening, 21(1), 74-87.

Figure 7 Characterization of equilibrium binding of ML342.

Influence of binding protocols on the observed affinity between ML342 and AM2.2. The cells were exposed to (1) 0.3 nM to 100 µM ML342 60 min before (closed triangle, Ki: 0.63 nM) the addition of 250 nM TO1-2p, (2) a mixture of ML342 and TO1-2p (filled circle, Ki: 0.35 µM), and (3) 250 nM TO1-2p 60 min before the addition of ML342 (closed diamond, Ki: 0.76 µM).

Wu, Y., Stauffer, S. R., Stanfield, R. L., Tapia, P. H., Ursu, O., Fisher, G. W., ... & Sklar, L. A. (2016). Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair. Journal of Biomolecular Screening, 21(1), 74-87.