Selection of Affibody Molecules Using Phage Display
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Updated: Aug 14, 2026

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits
Published on: February 16, 2014
Linnea Charlotta Hjelm1, Charles Dahlsson Leitao1, Stefan Ståhl1
1Department of Protein Science, KTH Royal Institute of Technology, 106 91 Stockholm, Sweden.
Abstract:
Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library. This is then followed by amplification of the library, which can then be used for biopanning using multiple methods. This protocol describes amplification of affibody libraries, followed by biopanning using phage display and analysis of the selection output. The general procedure is mainly for selection of first-generation affibody molecules from large naive (unbiased) libraries, typically yielding affibody hits with affinities in the low nanomolar range. For selection from affinity maturation libraries with the aim of isolating variants of even higher affinities, the procedure is similar, but parameters such as target concentration and washing are adjusted to achieve the proper stringency.
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