Journal: Bioconjugate chemistry

Article Title: Synthetic Tuning of Domain Stoichiometry in Nanobody–Enzyme Megamolecules

doi: 10.1021/acs.bioconjchem.0c00578

Figure Lengend Snippet: Characterization of megamolecule binding. A,B. Synthetic schemes of fluorescent megamolecules with either A, one nanobody domain (1N:1GFP); or B, two nanobody domains (2N:1GFP) conjugated to a fusion protein containing a sfGFP domain. C. Adherent culture microscopy of BT-474 cells incubated with 10 nM 1N:1GFP after 2 h. Scale bars are 200 μm (top) and 20 μm (bottom). D,E. Spheroid culture confocal microscopy of BT-474 cells incubated with 50 nM 1N:1GFP. Scale bar is 200 μm. D. Image of spheroids after 2 h incubation with 1N:1GFP. E. Single Z-stack slice of spheroid center after a 1 h incubation with 1N:1GFP. F. Averages of fluorescence mean pixel intensity (MPI) of fixed adherent cells incubated with 1N:1GFP (blue) or 2N:1GFP (red) over time. After 80 min, the wells were washed three times and imaged to track dissociation from the cells. Curves for association (kon) and dissociation (kcoff) were fit from nonlinear fit kinetics software packages using GraphPad Prism from 7 to 9 biological replicates. Error reported as standard deviation.

Article Snippet: Curves for association ( k on ) and dissociation ( k coff ) were fit from nonlinear fit kinetics software packages using GraphPad Prism from 7 to 9 biological replicates.

Techniques: Binding Assay, Microscopy, Incubation, Confocal Microscopy, Fluorescence, Software, Standard Deviation