Peptides as Baits for the Coprecipitation of SH2 Domain-Containing Proteins.

Azin Kiani, Pablo Rios, Maja Köhn

Methods in molecular biology (Clifton, N.J.) 2023

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Phosphotyrosine (pTyr)-containing amino acid sequences have regulatory effects on proteins that contain pTyr recognition motifs, such as Src Homology 2 (SH2) domains. Using pTyr-containing peptides as a bait for coprecipitation, by immobilization of the synthesized phosphopeptides to beads and incubation with cell lysates, enables to study the binding preference of the SH2 domain for the specific pTyr-sequence obtained from a pTyr-containing protein in a complex biological environment. Using phosphopeptides allows to not only assess the wild-type sequence, but also peptides that can contain modified sequences which carry a nonhydrolyzable pTyr or other modifications varying the binding strength and selectivity, for example, to create strong SH2 domain binders to inhibit their interaction with pTyr-containing proteins. This pulldown experiment can be used as an assay to evaluate the ability of a peptide to bind to the protein of interest in the cell lysate or investigate the selectivity of the peptide. Therefore, immobilizing phosphopeptides and using them as a pulldown tool has a wide range of applications. © 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Citation

Azin Kiani, Pablo Rios, Maja Köhn. Peptides as Baits for the Coprecipitation of SH2 Domain-Containing Proteins. Methods in molecular biology (Clifton, N.J.). 2023;2705:359-369