Citrate synthase activity (Sigma CS0720, 96-well)

Measure citrate synthase activity, a proxy for mitochondrial density, in coral host and symbiont fractions as the DTNB reaction rate at 412 nm, normalized to protein.

Version 1.0 · last updated 2026-10-05 · Used in 2 notebook entries ↓

At a glance

Input	Host or symbiont fraction (or a bead-beaten polyp) in lysis buffer, with known protein content
Output	Citrate synthase activity (mU mg⁻¹ protein)
Kit	Sigma-Aldrich Citrate Synthase Assay Kit CS0720 (manual) + Pierce BCA kit
Time	~1 h for one plate after protein is known (estimate)
Tested on	Astrangia poculata (symbiotic and aposymbiotic), host and symbiont fractions

Citrate synthase turns acetyl-CoA and oxaloacetate (OAA) into citrate and releases CoA-SH. CoA-SH reacts with DTNB to give a yellow product, measured at 412 nm. The rate measured before OAA is added (baseline) is subtracted from the rate after.

Materials

• Sigma CS0720 kit: 5× assay buffer, acetyl-CoA, OAA, DTNB, citrate synthase (positive control)
• Pierce BCA Protein Assay Kit (Total protein protocol)
• 100% ethanol (to dissolve DTNB), DI water
• Lysis buffer (below); bead tubes with 0.1 and 0.5 mm beads (whole polyps)
• 96-well flat-bottom plate, plate reader with kinetic mode at 412 nm, multichannel pipette

Solutions to prepare

Solution	Recipe	Storage
Lysis buffer (25 mM Tris pH 7.8, 1 mM EDTA, 10% glycerol)	100 mL: 2.5 mL 1 M Tris + 0.2 mL 0.5 M EDTA + 10 mL glycerol + 87.3 mL DI water	Room temp
1× assay buffer	1 part 5× buffer + 4 parts DI water (e.g. 0.7 mL + 2.8 mL). Make enough for the master mix + OAA + control	Make fresh
Acetyl-CoA, 30 mM	Dissolve the vial in 1 mL DI water	200 µL aliquots, −20 °C
DTNB, 10 mM	Dissolve the vial in 1 mL 100% ethanol	200 µL aliquots, −20 °C
OAA, 10 mM	1.3 mg OAA in 1 mL 1× assay buffer	−20 °C, max 1 week
Positive control	2 µL citrate synthase + 60 µL 1× assay buffer	On ice, same day

Procedure

A. Samples and protein

1. Fractions: use host and symbiont aliquots from Part C of the tissue protocol. Whole polyps: put a polyp in a bead tube with 500 µL lysis buffer, vortex ~2 min, spin 16,000 × g for 1.5 min and keep the supernatant.
2. Measure protein with BCA on a small aliquot.
3. Dilute each sample so that 20 µL contains 2–6 µg protein (Hawkins et al. 2016). Samples under 1 µg per 20 µL are too dilute to use.
   • e.g. 0.774 mg mL⁻¹ × 20 µL = 15.5 µg → dilute 3× (6.6 µL sample + 13.4 µL water).
4. Prepare 60 µL per sample (3 replicate wells × 20 µL). Negative control: 60 µL lysis buffer. Positive control: the diluted citrate synthase.

B. Reaction

5. Make the master mix: 170 µL per well:

   Per well	Final conc.
   3.3 µL 30 mM acetyl-CoA	~0.59 mM
   5 µL 10 mM DTNB	~0.29 mM
   161.7 µL 1× assay buffer

   Make ~10–25% extra (e.g. 15 wells’ worth for 12 wells).

6. Add 510 µL master mix (170 µL × 3) to each 60 µL sample/control tube and vortex.

7. Pipette 190 µL into each of 3 wells.

8. Baseline: kinetic read at 412 nm for ~3 min (e.g. 4 reads, ~49 s apart).

9. Add 10 µL 10 mM OAA to each well with a multichannel so all reactions start together. Pipette-mix.

10. Read again with the same kinetic settings.

C. Calculate

11. Rate (ΔA412 min⁻¹) after OAA minus the baseline rate, minus the negative control.
12. Convert to activity with the DTNB extinction coefficient and path length as in the kit manual. Normalize to the µg protein in the well and report as mU mg⁻¹ protein. Example R scripts: assay test, Astrangia nutrition.

Expected results

Sample	Activity
A. poculata symbiotic polyp	68.9 mU mg⁻¹
A. poculata aposymbiotic polyp	101.5 mU mg⁻¹

These are from the 2019 test and fall in the range reported for Exaiptasia pallida (Hawkins et al. 2016).

Troubleshooting

Problem	Likely cause	Fix
No increase after OAA	OAA degraded (unstable once dissolved)	Make OAA fresh, and don’t keep it more than a week at −20 °C
Reactions in the first columns run ahead	Staggered OAA addition	Add OAA with a multichannel and start reading immediately
Too many samples for the kit volumes	One kit covers limited wells	Scale the master mix; 150 wells needed ~24 mL 1× buffer (2019-05-15)

References

• Sigma-Aldrich CS0720 technical bulletin (PDF)
• Hawkins TD et al. 2016. PMC4834350
• Spinazzi M et al. 2012. Nature Protocols (PubMed 22653162)

Notebook entries using this protocol

Changelog

Date	Version	Change
2026-10-05	1.0	Rewritten from the 2019 assay development and Astrangia nutrition runs
2019-05	–	Host and symbiont fractions, 150-well plate (Astrangia nutrition)
2019-03	–	Assay developed on whole A. poculata polyps