Human Anti-MG Recombinant Antibody (clone L5*) (CAT#: PABZ-156)

Figure 1 Characterization of L5*/MG complex formation.

(a) Normalized fluorescence of L5* ternary complex and dL5* binary complex. Main (X) and ancillary (Y) excitation bands are marked. (b) Equilibria of dL5* binary complex. Plot shows proportion of 150 pM dimer protein assembled into a binary complex as a function of MG concentration. Ligand depletion analysis gives a K_D of 90 pM. (c) Equilibria of L5* ternary complexes. Plot of experimental data shows proportion of total V_L assembled into V_L/MG/V_L ternary complex (θ_V^VMV) as a function of MG concentration employing indicated initial V_L concentrations ([V]₀, continuous lines). Data are corrected for inner filter effect. Full assembly occurs over MG range of 25–200 μM for all initial V_L concentrations (linear plot of [MG], inset). Computed values of the proportion of monomer assembled into complex are shown for indicated V_L concentrations [V]₀ using K_d = 50 μM and cooperativity parameter α = 2 × 10⁵ (broken lines) or K_d = 1200 μM and α = 1 × 10⁸ (dotted lines). The shown equation predicts the maximum extent of ternary complex formation (θV^VMV_max) as a function of these two variables. (d) Isothermal titration calorimetry of L5* and dL5*. Listed thermodynamic parameters of dL5* (kJ/mol) are derived from these ∆H values and the experimentally determined K_d value in (b).

Szent-Gyorgyi, C., Stanfield, R. L., Andreko, S., Dempsey, A., Ahmed, M., Capek, S., ... & Bruchez, M. P. (2013). Malachite green mediates homodimerization of antibody V L domains to form a fluorescent ternary complex with singular symmetric interfaces. Journal of molecular biology, 425(22), 4595-4613.

Figure 2 VLVL dimer improves signal-to-noise and sensitivity.

(a) Confocal microscopy of dL5** displayed on yeast surface. Yeast cells expressing dL5** encoded by single copy chromosomal integrant were imaged on Zeiss LSM510 laser confocal microscope with identical instrument settings using 10–100,000 pM MG. To facilitate viewing, we adjusted image contrast using Zeiss software and inverted grayscale image. A representative quantitation is shown in Fig. S9. (b) Flow cytometry quantitation of dL5** displayed on yeast. Plot shows mean fluorescence values (640 nm excitation) of cells treated with MG over the indicated concentration range. Cellular autofluorescence in the absence of dye is about 6.

Szent-Gyorgyi, C., Stanfield, R. L., Andreko, S., Dempsey, A., Ahmed, M., Capek, S., ... & Bruchez, M. P. (2013). Malachite green mediates homodimerization of antibody V L domains to form a fluorescent ternary complex with singular symmetric interfaces. Journal of molecular biology, 425(22), 4595-4613.