Abstract
In eukaryotes, mitogen-activated protein kinases (MAPKs) are one of the best studied pathways for posttranslational modification-mediated regulation of protein functions. Here, we describe a rapid in vitro method to screen potential protein phosphorylation sites targeted by MAPKs. The method is based on PCR-mediated mutagenesis together with a type IIs restriction digest. Screening for the successfully mutated clones is further facilitated through introduction of a second diagnostic restriction site. Besides time-saving, this reduces the cost for sequencing confirmation of the positive clones, which are used for subsequent recombinant protein production and kinase assay validation.
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Acknowledgement
M.A.T.P-.F. and L.E.-L. are supported by the Saxony-Anhalt Excellence Network Graduate program (W21040908) and the ProNET-T3 program (03ISO2211B), respectively. MAPK signaling work is financed by the Collaborative Research Center program, SFB648.
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Eschen-Lippold, L., Bauer, N., Löhr, J., Palm-Forster, M.A.T., Lee, J. (2014). Rapid Mutagenesis-Based Analysis of Phosphorylation Sites in Mitogen-Activated Protein Kinase Substrates. In: Komis, G., Šamaj, J. (eds) Plant MAP Kinases. Methods in Molecular Biology, vol 1171. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0922-3_15
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DOI: https://doi.org/10.1007/978-1-4939-0922-3_15
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-0922-3
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