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

Protokoll

1. Colorimetric Citrate Synthase Activity Assay for D. melanogaster

1. Collect ten adult flies for each sample. Collect at least triplicate samples for each genotype.
2. 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.
3. 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 thoraxes by a pair of forceps, and then isolate the fly abdomens by cutting along the border of the thorax and abdomen using a pair of scissors. Collect the fly thoraxes for the muscle citrate synthase activity assay.
   NOTE: Determine the fresh weight of tissue at this step if using it to normalize citrate synthase activity.
4. Transfer 10 adult fly thoraxes to 100 µL of ice-cold extraction buffer immediately and homogenize with a pestle on ice. To keep the samples ice-cold, homogenize the samples for 5-10 s on ice, then have the samples sit on ice for 5 s, and repeat until all the tissues in the tube are homogenized completely.
   NOTE: Tape the tube, check the homogenates to make sure that there are no clots in the homogenates and that the tissues in the tube are homogenized completely. All sample treatments should be performed on ice.
5. Take 10 µL of each homogenized sample in a new tube for protein content measurement. Keep the samples for protein measurement on ice.
   NOTE: The protein samples can be frozen and stored at -80 °C for later analysis. The protein concentrations serve as an internal parameter for normalization of citrate synthase activities.
6. Add 400 µL of ice-cold extraction buffer to each remaining sample to make a total volume of 500 µL. Mix well by gently pipetting up and down at least 5x, avoiding bubble formation. For each reaction, add 1 µL of diluted cell lysate to 150 µL of the freshly prepared reaction solution (20 mM Tris-HCl (pH 8.0), 0.1 mM DTNB, 0.3 mM acetyl CoA, 1 mM oxaloacetic acid). Mix thoroughly and immediately by gently pipetting, avoiding bubble formation. Measure the absorbance at 412 nm every 10-30 s for 4 min at 25 °C using a plate reader capable of measuring absorbance at 412 nm at minimum intervals of 10 s.
   NOTE: Change the tip before pipetting a different reagent and avoid forming bubbles in the wells. Incomplete mixing of the reagents may cause variations in the measurements. The citrate synthase activity assay should be done at room temperature immediately after the samples are collected, as this assay is used to quantify intact mitochondrial mass. The reaction buffer should be prepared immediately before use and should not be stored.
7. Plot data as optical density (OD) absorbance (abs) (Y-axis) versus time (in minutes) (X-axis). Then calculate the slope for the linear portion of the curve, where the reaction rate is
   
   Divide the value by the sample protein concentration to normalize the citrate synthase activity. The citrate synthase activity is calculated as
   
   NOTE: The sample protein concentration can be measured by a protein concentration assay kit.

Materialien

Name	Company	Catalog Number	Comments
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