Binding assay assessment of the quality of the libraries, and the sequences selected. (A) The initial library (R0) and Round 10 library (R10) was prepared by the cDNA display method (up to step 4, Fig. 1A). The R10 library was reduced with dithiothreitol (DTT) to obtain the R10-D library. All three forms were incubated with non-coated beads, IL-6R coated beads or AChBP coated beads, washed and detected with Penta.His HRP (see Methods). (B) Sequences of the selected molecules compared to the native 3F sequence (shown as 'wt'). The loop sequences that were randomized are shown as letters and the non-randomized β-sheet residues are shown as asterisks. The cysteine residues are shown in red (C42 and C53 are indicated by arrows). '% Population' indicates the percentage of selected molecules out of the 30 clones that were sequenced. For example, approximately 70% of the R10-17-like proteins were selected. The sequences were assigned to respective groups (such as R10-17-like) based on the mutations observed in the loops (shown in italics). Similar loop sequences are shown in the same color. Mutations in the non-randomized portions are indicated by the mutated residues. C42 and I44 were found to be mutated to Y/H and T residues, respectively.