Document Type

Article

Publication Date

2018

DOI

10.3791/58547

Publication Title

Journal of Visualized Experiments: Immunology and Infection

Issue

141

Pages

e58547 (1-8)

Abstract

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of γ-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

Rights

Copyright © 2018 Journal of Visualized Experiments.

Included with the kind written permission of the lead author and the publisher.

Comments

A corresponding video article is available on the publisher's website exclusively.

Original Publication Citation

Pokhrel, A., Poudel, A., & Purcell, E. B. (2018). A purification and in vitro activity assay for a (p)ppGpp synthetase from Clostridium difficile. Journal of Visualized Experiments: Immunology and Infection (141), Article e58547. https://doi.org/10.3791/58547

ORCID

0000-0002-8736-0433 (Purcell)

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