Phage panning

Cell Culture and Phage Panning (Methods Outlined in Subheadings 3.1 and 3.2)... 

Approximately 15 min before the end of the clearing step, prepare the phage panning solution as follows ... 

Furuta, M., Ito, T., Eguchi, C., Tanaka, T., Wakabayashi-Takai, E., Kaneko, K. (2002). Two-dimensional electrophoresis/phage panning (2D-PP) a novel technology for direct antibody selection on 2-D blots. J Biochem 132, 245-251. 

Most of die sequences of the kallikrein binders revealed consensus sequences in one or the other of the peptide loops. Tliree new libraries were created widi each one of die three consensus regions in one loop and six random amino acids in the odier loop (Fig. 4a). Tlie libraries were mixed and phage panned under stringent conditions (1 nM to 200 pM biotinylated kallikrein). Tlie random sequence converged to a new consensus, yielding clones widi consensus sequences in both loops (Fig. 4b). Inhibidon assays revealed that tlie IC50 of die best inhibitor (PK15) was 20 nM when tested as a D1-D2 fusion protein. 

Use 100-200 pL of this purified phage and 400 pL of TBST for next panning. 

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