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The data were described using a one-site ligand binding model, with a ligand concentration of 0.19 μmol site mg−1 C, and a log K′ of 6.2.
Fortunately, the Kd value could be determined at a lower temperature; at 10°C, the heat exhausted could be fitted to a one-site binding model with a Kd value of 21.0 ± 2.1 µmol/L (Fig. 1C).
However, the amide cross-peak of Leu31 demonstrates a significant urea dependence that can be fitted to a two-state binding model with a dissociation constant of 0.95 ± 0.04 M.
Both datasets could be fit well to a three-site binding model with a single affinity for Nck.
The data could be fitted with a one-site competition binding model with a Ki of 1.4 μM, showing that the USP4 insert competes with Ub for binding to USP4 D1D2.
The data could only be reliably fit using a one-site binding model with a Kd = 23 ± 4 μM and a JAZ ZF1/RNA stoichiometric ratio of 2 1.
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Docking simulation has been performed to position compound 3e into the EGFR active site to determine the probable binding model, with an estimated binding free energy value of −10.7 kcal/mol.
Although the binding data for the gα8 - cells appeared to show a sigmoidal curve, F tests comparing binding with a Hill coefficient of 1 to binding models with a variable Hill coefficient indicated that a model with a Hill coefficient of 1 was preferred for all cell lines except for gα5 -, where a model with a Hill coefficient of 2.9 ± 0.9 (mean ± s.e.m, n = 3) was preferred.
The final values for the stoichiometry of interaction (N = 0.9) are consistent with a 1 1 binding model with mid-nanomolar affinity given the potential error in protein concentration estimates.
The data were fitted with a nonlinear least squares routine using a single-site binding model with Origin for ITC v.5.0 (Microcal).
Analysis with a two-site binding model with the assumption that the binding at each site generates the same change in CD signal improved the χ value to 1.2.
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