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. 2012 Feb 20;7(2):e31298. doi: 10.1371/journal.pone.0031298

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Hoffmann et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Specificity of ubiquitin variant 10F. The interaction of the purified, His₆-tagged binder with different immobilized target proteins (500 ng per well, human serum was used without dilution) is shown. (NGF: nerve growth factor, nPAC-1Rs: short splice form of N-terminal domain of human PACAP-receptor 1) (B) Characterization of the specificity by competition of the interaction of TNF-alpha and 10F. Biotinylated TNF-alpha (1 µM), preincubated for 1 h with competitor (etanercept: TNF-receptor 2 dimerized by fusion to IgG F_c), was incubated with immobilized 10F (100 ng per well). Detection by avidin-peroxidase demonstrates competition by etanercept. (C) Concentration-dependent competition ELISA. 10F (100 nM, His₆-tagged), preincubated with different concentrations of TNF-alpha, was incubated with immobilized TNF-alpha (150 ng per well). An IC₅₀ value of 289±53 nM was determined.