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Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins
JoVE Journal
Chemistry
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JoVE Journal Chemistry
Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

DOI:
10.3791/52142-v

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07:25 min

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October 17, 2014

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, , ,
1School of Life Sciences,University of Nottingham

Chapters

  • 00:05Title
  • 01:13Measurement of Association and Dissociation Rate Constants for mantATP
  • 04:18Measurement of the Dissociation Rate Constant for mantADP
  • 05:19Results: Analysis of the ATP Turnover Cycle of Kinesins
  • 06:45Conclusion

Summary

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Kinesins are characterized by nucleotide-dependent interaction with microtubules: a cycle of ATP turnover coupled to a cycle of microtubule interaction. Here, we describe protocols to analyze the kinetics of individual nucleotide transitions in the ATP turnover cycle of a kinesin using fluorescently labeled nucleotides and stopped-flow fluorescence.

Tags

Keywords: KinesinATP TurnoverMicrotubuleStopped-flow FluorescenceFluorescently Labeled NucleotidesNucleotide Transition KineticsRapid MixingReaction Kinetics

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