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Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation
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JoVE Journal Chemistry
Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

DOI:
10.3791/53529-v

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10:52 min

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January 06, 2016

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, ,
1Department of Chemistry and Biology, Program in Molecular Science,Ryerson University

Chapters

  • 00:05Title
  • 00:58Yeast Cell Culture and Radiolabeling of Phosphoinositides
  • 02:58Deacylation and Extraction of the Radiolabeled Phosphoinositides
  • 05:14Separation of Tritium-labeled Glycero-inositides by High Pressure Liquid Chromatography (HPLC)
  • 07:15Data Analysis
  • 08:55Results: HPLC Analysis of Radiolabeled Phosphoinositides
  • 09:55Conclusion

Summary

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Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Tags

Keywords: RadiolabelingPhosphoinositidesHPLCFlow ScintillationYeastMammalian CellsInositolPerchloric AcidEDTACentrifugationSonicationLiquid CultureCell GrowthCell Lysis

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