Magnetic beads handler

Four rounds of biopanning using Fab Library E (kind gift of A. Koide) were performed as follows using a protocol adapted from previous studies 41, 42 . The first round of selection was performed manually using 300 nM of biotinylated K29-linked diubiquitin immobilized onto 250 µL of Streptavidin Paramagnetic Particles (Promega). Following washing with Selection Buffer (SB; 20 mM Hepes, pH 7.5, 150 mM NaCl, 0.5% BSA, 0.05% Tween-20), the beads were incubated with Library E for one hour at room temperature (RT). The beads were then washed three times with SB and used to directly infect log-phase E. coli XL-1 blue cells. Following infection, 2XYT media supplemented with ampicillin and M13 helper phage was added and phage were amplified overnight. To increase the stringency of selection, three additional rounds of sorting were performed in the presence of 1.5-µM of mono-ubiquitin as a competitor in solution. Further, the concentration of K29-linked diubiquitin was reduced each round as follows: 150 nM in round 2, 75 nM in round 3, and 15 nM in round 4. These three rounds were performed at RT semi-automatically using a Kingfisher magnetic beads handler. The overnight-amplified elution pool from each preceding round of selection was used as the input for the following round of sorting.