Application Notes

Application Note-Cube-MOX-Affinity Measurements Without Disrupting Protein Conformation_002

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9 A P P L I C A T I O N N O T E Figure 6: (A) DLS analysis of copolymer nanodisc-stabilized GLP1R. The cumulant analysis reported the polydisper- sity index (PDI) of the sample to be 0.06 ± 0.02. The size distribution analysis reported the hydrodynamic radius (rH) of the GLP1R nanodisc to be 5.51 ± 0.12 nm. (B). Binding of Exendin (9-39) to nanodisc-stabilized GLP1R. The fluo- rescently labeled GLP1R was used at a constant concentration of 20 nM with varying concentrations of the Exendin (9-39) in 20 mM HEPES pH 7, 150 mM NaCl, and 0.005% Tween 20. Ratios of the fluorescence intensities at 670 and 650 nm were used for interaction analysis yielding the Kd of 4.51 nM with S/N ratio of 10.1. Error bars represent the standard deviation of 3 independent measurements and with a confidence of 95%, Kd is within 1.79-5.91 nM. The copolymer nanodisc-stabilized full-length GPCR, GLP1R, was found to be highly mon- odisperse and thermally stable. Using this high-quality protein sample, the Spectral Shi measurement was successfully employed to study the binding of a peptide to the membrane nanodiscs in the assay buffer without further optimization steps. The Monolith X is designed for user-friendly operation, offering a straightforward assay setup and intuitive so ware that provides detailed guidance to users. This ease of use makes interaction analysis ac- cessible to interdisciplinary scientists and enables biophysical experts to be more efficient, especially when working with challenging protein targets. 0 2 4 6 8 10 12 14 16 Relative probability 1 10 100 1000 Radius [nm] 0.88 0.9 0.92 0.94 0.96 0.98 1 1.02 670nm / 650nm 1x10 -11 1x10 -10 1x10 -9 1x10 -8 1x10 -7 1x10 -6 1x10 -5 Ligand Concentration [M] (A) (B)

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