To compare the binding affinity of Fn3 clone 1.2 for different PFO mutants, EBY100 yeast were transformed with 1.2 using the EZ Yeast transformation kit (Zymo Research, Irvine, CA) and induced to display the Fn3 as previously described.28 Soluble PFO clones were labeled with Alexa Fluor 647 following the manufacturer’s instructions (Life Technologies). 0.3 million yeast displaying 1.2 were incubated with 500 nM PFO for 30 min at 4 °C and analyzed on an Accuri C6 cytometer (BD Accuri Cytometers, Ann Arbor, MI). The measured fluorescence intensities were normalized to that of PFO.
To analyze the binding specificity of the C225.2/PFO complex for cell lines of interests, fluorescently labeled PFO clones were preincubated with C225.2 or the control sm3e.2 at an equimolar ratio of PFO to its binding site, for 30 min at 4 °C. The PFO mixtures were then diluted in PBSA to the desired concentrations, incubated with A431 or CHO-K1 cells for 1.5 h at 4 °C in suspension, and analyzed on the Accuri C6 cytometer (BD Accuri Cytometers). Fluorescence intensities were normalized to the maximal value obtained for each PFO clone.
To analyze the binding specificity of the C225.2/PFO complex for cell lines of interests, fluorescently labeled PFO clones were preincubated with C225.2 or the control sm3e.2 at an equimolar ratio of PFO to its binding site, for 30 min at 4 °C. The PFO mixtures were then diluted in PBSA to the desired concentrations, incubated with A431 or CHO-K1 cells for 1.5 h at 4 °C in suspension, and analyzed on the Accuri C6 cytometer (BD Accuri Cytometers). Fluorescence intensities were normalized to the maximal value obtained for each PFO clone.