Abstract
Phosphorylation assay is a widespread technique usually necessary for the identification of a specific kinase substrate and/or for the measurement of kinase activity. As an example of the technique, here we describe an assay aimed to test the phosphorylation of the myelin basic protein (MBP) by protein kinase C (PKC), which is overexpressed and purified from Neurospora. The kinase is immunopurified from Neurospora using the expression vector pMYX2 and the FLAG epitope. The purified PKC and the MBP are then incubated in the presence of radioactive ATP, and the phosphorylated product is separated using the polyacrylamide gel electrophoresis technique.
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References
Liu, Y. (2003) Molecular mechanisms of entrainment in the Neurospora circadian clock. J. Biol. Rhythms 180, 195–205.
Vogel, H. J. (1956) A convenient growth medium for Neurospora. Microb. Genet. Bull. 13, 42–43.
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© 2007 Humana Press Inc.
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Franchi, L., Macino, G. (2007). In Vitro Phosphorylation and Kinase Assays in Neurospora crassa . In: Rosato, E. (eds) Circadian Rhythms. Methods in Molecular Biology™, vol 362. Humana Press. https://doi.org/10.1007/978-1-59745-257-1_32
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DOI: https://doi.org/10.1007/978-1-59745-257-1_32
Publisher Name: Humana Press
Print ISBN: 978-1-58829-417-3
Online ISBN: 978-1-59745-257-1
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