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A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

DOI :

10.3791/58547-v

November 3rd, 2018

November 3rd, 2018

8,067 Views

1Department of Chemistry and Biochemistry, Old Dominion University

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

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