Abstract
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.
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Kiani, A., Rios, P., Köhn, M. (2023). Peptides as Baits for the Coprecipitation of SH2 Domain-Containing Proteins. In: Carlomagno, T., Köhn, M. (eds) SH2 Domains. Methods in Molecular Biology, vol 2705. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3393-9_19
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DOI: https://doi.org/10.1007/978-1-0716-3393-9_19
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3392-2
Online ISBN: 978-1-0716-3393-9
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