Figure 1: ELISA analysis of D1 variants after two rounds of error-prone PCR followed by selection using ribosome display.
DARPins at a 30 nM concentration were incubated in 10 nM biotinylated peptide-coupled tubulin-coated wells. For off-rate estimation, after DARPin binding and the washing steps, the wells were incubated either with buffer or with a 100 nM tubulin solution, to prevent the rebinding of detached DARPins to immobilized tubulin. The DARPins were ranked as low-, medium- or high-affinity binders. D1 is the parent DARPin whereas off7 is an unrelated one8, not binding to tubulin and used as a negative control. Error bars are standard deviations from duplicate experiments. AU, absorbance unit.
