title: A protocol for phage display and affinity selection using recombinant protein baits.
abstract: Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.
authors.affiliation: Department of Horticulture, University of Kentucky.
authors.forename: Rekha
authors.initials: R
authors.lastname: Kushwaha
authors.affiliation: Department of Horticulture, University of Kentucky.
authors.forename: Kim R
authors.initials: KR
authors.lastname: Schäfermeyer
authors.affiliation: Department of Horticulture, University of Kentucky ; adownie@uky.edu.
authors.forename: A Bruce
authors.initials: AB
authors.lastname: Downie
mesh_terms: D061505:Cell Surface Display Techniques / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D055767:Immobilized Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D055767:Immobilized Proteins; D011994:Recombinant Proteins