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Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay
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Bioquímica
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JoVE Journal Bioquímica
Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

DOI:
10.3791/55129-v

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09:17 min

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January 11, 2017

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, ,
1Department of Chemistry,Stony Brook University, 2Department of Physiology and Biophysics,Stony Brook University

Capítulos

  • 00:05Título
  • 00:53Setting up the 96-well Dot Blot Apparatus
  • 02:02Reaction Initiation and Quenching
  • 02:57Spotting of Quenched Reactions on Nitrocellulose
  • 04:51Nitrocellulose Processing
  • 05:48Preparing Nitrocellulose Membrane for Scintillation Counting
  • 07:31Results: Quantification of the Autophosphorylation of V. parahaemolyticus H-NOX Associated Histidine Kinase (Vp1876)
  • 08:35Conclusion

Summary

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We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

Tags

Bacterial Histidine KinaseAutophosphorylationQuantificationNitrocellulose Binding AssayTwo-component SignalingPost-translational ModificationRadioactive LigandsHigh-throughputKinetic ParametersProtein-protein InteractionsFiltration96-well Dot-plot ApparatusGamma-32P ATPPhosphoric Acid

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