Figure 1

Panning procedures. (a) Schematic representation of a traditional biopanning procedure. After target incubation (I) and postcoating, a phage library is added and incubated (II). In a washing step (III) non-bound phages are washed away followed by an elution step (IV). The E. coli cells are consequently infected with the eluted phages (V) and out-plated on agar plates for amplification. After overnight grow the cells are harvested and the phages revovered by a precipitation step (VI). These phages are used for subsequent panning rounds until high affinity phages are obtained. Schematic representation of the Chromato-panning procedure: (b) On-column panning. A cryogel column with the target protein covalently coupled, is washed with PBS buffer followed by loading of a sample of the phage library at 0,5 ml.min^-1. After washing out the non-bound phages, the bound phages can be eluted with 1 M NaCl and further used for infection of E. coli cells as described for a classical panning procedure. (c) On-column infection. After the on-column panning as in (b), without elution of bound phages, the column is incubated at 37°C and one column volume of E. coli cells is applied on the column, followed by an incubation time (no flow) for 45 min. Next, the infected E. coli cells are eluted and out-plated on agar plates. After overnight grow, the cells are harvested and the phages recovered by a precipitation step. (d) Evaluation of the elution solvent in biopanning. Two similar chromato-panning procedures were performed with a linear hexamer peptide library using a different elution solvent. The bound phages were eluted from the column using either 0.1 M glycine, pH 2.2 (open square) or 1 M NaCl (black square) as elution solvent. The eluted phages were used to infect E. coli cells. After amplification and harvesting, the binding affinity of the harvested phages was analyzed using phage ELISA as described in Materials and Methods.