Accedi

Adelphi University

2 ARTICLES PUBLISHED IN JoVE

Developmental Biology

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

¹Biology Department, Adelphi University, ²Department of Ecology and Evolutionary Biology, Brown University

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

Chemistry

NMR-Based Activity Assays for Determining Compound Inhibition, IC₅₀ Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

Brian J. Stockman¹, Abinash Kaur¹, Julia K. Persaud¹, Maham Mahmood¹, Samantha F. Thuilot¹, Melissa B. Emilcar¹, Madison Canestrari¹, Juliana A. Gonzalez¹, Shannon Auletta¹, Vital Sapojnikov¹, Wagma Caravan^1,2, Samantha N. Muellers^1,3

¹Department of Chemistry, Adelphi University, ²Department of Chemistry, Washington University in St. Louis, ³Department of Chemistry, Boston University

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 μM and 250 μM, dose-response assays for determining IC₅₀ values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.