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In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease
JoVE Journal
Medicine
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JoVE Journal Medicine
In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease
DOI:

10:16 min

December 20, 2017

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Chapters

  • 00:05Title
  • 00:35Site-directed Mutagenesis
  • 01:45Transformation and Screening for the Desired Clone
  • 02:35pcDNA3.1/GLA and pcDNA3.1/GAA Plasmid Transfection and Treatment of HEK293H
  • 04:00Cell Harvest
  • 04:43Homogenization by Freezing and Thawing and BCA Assay
  • 06:17Enzyme Activity Measurement with Artificial 4-Methylumbeliferyl Substrate
  • 08:19Results: Enzyme Activity Measurement Mutagenized 3 α-galactosidase and 3 Acid α-glucosidase
  • 09:35Conclusion

Summary

Automatic Translation

There is a demand to make pre-clinical testing for a novel class of "orphan" drugs called pharmacological chaperones reproducible, fast, and efficient. We developed a simple, highly standardized, and versatile cell culture-based assay to screen for eligible patients as well as novel pharmacological chaperone drugs.

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