HomeFAQsWhen performing an experiment using Ph.D.™ Phage Display, the ELISA indicates that background binding to the plate is as high as binding to the target.
FAQ: When performing an experiment using Ph.D.™ Phage Display, the ELISA indicates that background binding to the plate is as high as binding to the target.
If panning against a polystyrene plate coated with the target (direct coating method), it is possible to inadvertantly select peptides that specifically bind the polystyrene surface (see Adey, N. B. et al. (1995) Gene 156, 27-31). These peptides will yield identical ELISA signals in the presence and absence of target, since the ELISA plate is also made of polystyrene. Such "plastic binders" are typically rich in aromatic residues (Phe, Tyr, Trp, His), which often alternate (the sequence FHWTWYW is a plastic binder discovered and characterized at NEB). Selection of plastic binders often occurs in the absence of a strong target preference for peptide sequences present in the library: other libraries may yield the desired target specific sequences. Selection of polystyrene-specific peptides can be avoided by using the bead capture protocol described in the Manual. The phage is reacted with the target in solution, and the phage-target complexes are then captured onto beads that specifically bind the target (protein A-agarose for antibody targets, glutathione-agarose for GST fusions, etc.). Unbound phage is removed by extensively washing the beads in a microfuge tube. Unlike polystyrene, neither the beads (typically crosslinked agarose) nor the microfuge tube (polypropylene) are likely to select specific peptide sequences from the library, although the species conjugated to the beads (protein A, glutathione, etc.) might. To avoid selection of bead-specific ligands, we suggest either alternating rounds between different beads specific for the target (e.g. protein A beads for rounds 1 and 3, protein G beads for round 2 for antibody targets), or adding a subtractive panning step, beginning with round 2, in which the phage pool is first reacted with the beads alone (no target), the beads discarded, and the supernatant from this step reacted with the target.
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