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

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

DOI:
10.3791/58547-v

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09:53 min

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November 03, 2018

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, ,
1Department of Chemistry and Biochemistry,Old Dominion University

Chapters

  • 00:04Title
  • 00:44Protein Purification by Nickel Affinity Chromatography
  • 03:30Protein Activity Assay by Thin Layer Chromatography
  • 07:30Results: Thin Layer Chromatography Reveals that C. difficile RSH is an Active ppGpp Synthetase
  • 09:01Conclusion

Summary

Automatic Translation

Automatic Translation

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.

Tags

Keywords: Protein PurificationIn Vitro Activity Assay(p)ppGpp SynthetaseClostridium DifficilePhosphotransfer EnzymesEnzyme Substrate SpecificityReaction KineticsTLCSDS-PAGECoomassie Blue Staining

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