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Specific binding was obtained by subtracting non-specific binding from total binding.

The dissociation constant (KD) for the UIM-Ub binding was obtained by fitting the titration curves [61].

Specific binding was obtained by using 10 μM fluoxetine as a displacer.

For each sample, antigen-specific binding was obtained by subtracting the MFI for beads coated with GST alone from those of beads coated with GST VP1.

Specific binding was obtained by subtracting non-specific binding (in the presence of a 100-fold excess unlabeled EGF) from total binding.

Duplicates of membrane preparations equivalent to 0.1 mg protein were incubated with I-EGF (50 or 100 ng; Amersham Life Sciences, Arlington Heights, IL) in 200 μl reaction buffer (5 mM Tris-HCl, pH 7.0, 125 mM sucrose, 75 mM NaCl, 0.5 mM Ca2+ and 0.5% BSA) at 25°C for 60 min. Non-specific binding was obtained by adding 100× non-radioactive EGF (Sigma Biochemicals, St . Louis MO).

It is found that the optimal layers to obtain large reduction of protein adsorption and availability of functional groups for binding are obtained by using mixtures of flexible and rod-like molecules.

IC50 values (peptide concentration that gives 50% binding) were obtained by nonlinear least-squares fitting of the data to a four parameter sigmoidal equation (XLfit3, IDBS).

(1219) More-detailed information about structural requirements for binding and, indirectly, the geometry of the binding site was obtained by classification analysis, 1222) pharmacophore studies, pseudo-receptor modeling, 1225) GRIND descriptor studies, 1226) and 3D-QSAR analyses, which were combined with the homology models, based on the available bacterial ABC transporter structures.

The overall consensus binding motif was obtained by multiple alignments of the significant binding sequences with ClustalW, and it was visualized with SeqLogo program.

Similar to several previous studies [ 4, 10], for computing PCC, the binding value was obtained by 1 - log IC50)/ log 50, 000), where IC50 is binding affinity measured in nM.