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Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase
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Biochemistry
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JoVE Journal Biochemistry
Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

DOI:
10.3791/57798-v

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10:33 min

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October 15, 2018

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, , , ,
1Division of Pharmaceutical Sciences, School of Pharmacy,University of Missouri-Kansas City

Chapters

  • 00:04Title
  • 00:45Cloning and Purification of Untagged Human TET2 Dioxygenase
  • 05:305mC Oxidation by TET2 Dioxygenase
  • 07:02Quantitative Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)-based Assay Development
  • 08:31Results: Investigating the Role of the TET2 Enzyme in Normal Development and Disease
  • 09:26Conclusion

Summary

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Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

Tags

Keywords: TET25-Methylcytosine DioxygenaseProtein PurificationLC-MS/MSBacterial TransformationProtein ExpressionIPTG InductionE. Coli BL21 DE3EpigeneticsDemethylation

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