Figure 4. NMR analysis of the titration of 10F with TNF-alpha.

¹H-¹⁵N fHSQC spectra of ¹⁵N-labeled ubiquitin variant 10F in presence of increasing concentration of unlabeled TNF-alpha were recorded in PBS, 1 mM EDTA pH 7.4 with 0.05% Tween-20. The relative intensity decrease of two amid resonances was plotted against molar ratio of both proteins. As determined by the intersection point of the linear fits of the initial part and the plateau part of the curve 2.88±0.18 TNF-alpha monomers are bound per 10F molecule.