Citrate Synthase Activity Assay: A Colorimetric Assay to Measure Mitochondrial Citrate Synthase Activity in Drosophila Tissue Homogenate

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…

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