Citrate Synthase Activity Assay: A Colorimetric Assay to Measure Mitochondrial Citrate Synthase Activity in Drosophila Tissue Homogenate
Published: April 30, 2023
Abstract
Source: Wei, P., et al., A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster. J. Vis. Exp. (2020)
This video describes a colorimetric assay to measure the mitochondrial citrate synthase activity assay present in the tissue homogenate of Drosophila.
Protocol
1. Colorimetric Citrate Synthase Activity Assay for D. melanogaster Collect ten adult flies for each sample. Collect at least triplicate samples for each genotype. Prepare 500 µL of ice-cold extraction buffer containing 20 mM HEPES (pH = 7.2), 1 mM EDTA, and 0.1% triton X-100 in a 1.5 mL test tube for each sample. Anesthetize adult flies with CO2 on an anesthesia pad and isolate the desired tissues. To isolate the adult fly thoraxes, for example, fix the fly t…
開示
The authors have nothing to disclose.
Materials
| 2-[4-(2-Hydroxyethyl)-1- piperazinyl]ethanesulfonic acid (HEPES) | Sigma-Aldrich | V900477 | |
| 2-Amino-2-(hydroxymethyl)-1,3- propanediol (TRIZMA Base) | Sigma-Aldrich | V900483 | |
| Acetyl-CoA | Sigma-Aldrich | A2181 | |
| Dithio-bis-nitrobenzoic acid (DTNB) | Sigma-Aldrich | D8130 | |
| Ethylenediaminetetraacetic acid (EDTA) | Sigma-Aldrich | V900106 | |
| Oxaloacetate | Sigma-Aldrich | O4126 | |
| Pellet pestle | Sangon | F619072 | |
| Pellet pestle motor | Tiangen | OSE-Y10 | |
| Plate reader | BioTek | Eon | |
| Protein BCA Assay kit | Beyotime | P0010S | |
| Scissors | WPI | 14124 | |
| Triton X-100 | Sangon | A110694-0100 |
