Advertisement

In Vitro Phosphorylation and Kinase Assays in Neurospora crassa

  • Lisa Franchi
  • Giuseppe Macino
Part of the Methods in Molecular Biology™ book series (MIMB, volume 362)

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.

Key Words

Neurospora PKC protein expression phosphorylation kinase assay 

References

  1. 1.
    Liu, Y. (2003) Molecular mechanisms of entrainment in the Neurospora circadian clock. J. Biol. Rhythms 180, 195–205.CrossRefGoogle Scholar
  2. 2.
    Vogel, H. J. (1956) A convenient growth medium for Neurospora. Microb. Genet. Bull. 13, 42–43.Google Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Lisa Franchi
    • 1
  • Giuseppe Macino
    • 1
  1. 1.Dipartimento di Biotecnologie Cellulari ed EmatologiaRomeItaly

Personalised recommendations