Total antioxidant capacity (OxiSelect TAC assay)

Measure copper-reducing total antioxidant capacity of coral host tissue against a uric acid standard curve (Cell Biolabs OxiSelect STA-360).

Version 1.0 · last updated 2026-10-05 · Used in 1 notebook entries ↓

At a glance

Input	500 µL frozen airbrushed homogenate (host supernatant used)
Output	TAC in µM copper-reducing equivalents (uric acid equivalents), normalized to protein
Kit	Cell Biolabs OxiSelect Total Antioxidant Capacity Assay Kit, STA-360 (manual)
Time	~1 h for one plate (estimate)
Tested on	Porites astreoides adults

Antioxidants in the sample reduce Cu²⁺ to Cu⁺, which forms a coloured complex read at 490 nm. Absorbance is read before and after copper is added; the difference is compared to uric acid standards.

Materials

• OxiSelect TAC kit (reaction buffer, copper ion reagent, stop solution, uric acid)
• 1 M NaOH, 1× PBS, DI water
• Refrigerated centrifuge, 96-well plate, plate reader at 490 nm, orbital shaker, multichannel pipette

Solutions to prepare

Store prepared reagents at 4 °C for up to 3 months.

Solution	Recipe
1× reaction buffer (1:100)	400 µL reaction buffer + 39.6 mL 1× PBS
1× copper ion reagent (1:100)	1 mL copper ion reagent concentrate + 99 mL DI water
1× stop solution (1:10)	1.5 mL stop solution + 13.5 mL DI water
Uric acid, 2 mM	Dissolve 10 mg uric acid in 1 mL 1 M NaOH (≈ 60 mM), then 100 µL + 2.9 mL DI water

Make the standard curve by serial dilution of the 2 mM uric acid, following the kit manual:

Uric acid standard dilutions used in the 2019 run

Procedure

A. Samples

1. Thaw 500 µL homogenates on ice.
2. Spin 5,200 rpm, 5 min, 4 °C to pellet symbionts.
3. Split the supernatant: 100 µL for TAC, 400 µL for protein (Total protein). Keep on ice.

B. Assay

4. Add 20 µL standard or sample per well, in duplicate.
5. Add 180 µL 1× reaction buffer with a multichannel and pipette-mix.
6. Initial read at 490 nm.
7. Add 50 µL 1× copper ion reagent. Incubate 5 min at room temperature on an orbital shaker.
8. Add 50 µL 1× stop solution.
9. Final read at 490 nm.

C. Calculate

10. ΔA490 = final − initial for every well. Subtract the blank.
11. Fit the uric acid standard curve and calculate µM uric acid equivalents. Convert to µM copper-reducing equivalents (CRE) with the conversion factor given in the kit manual.
12. Normalize to protein from the same homogenate.

Plate layout

In the 2019 run, 8 standards in duplicate filled row A and the start of row B, leaving room for 40 samples in duplicate. Two of the kit’s standards were dropped to fit all samples on one plate (2019-11-26).

Troubleshooting

Problem	Likely cause	Fix
Uric acid won’t dissolve	Needs a basic solution	Dissolve in 1 M NaOH before diluting in water
Sample above the top standard	High antioxidant content	Dilute in 1× PBS and re-run

Notebook entries using this protocol

Changelog

Date	Version	Change
2026-10-05	1.0	Rewritten from the 2019 Porites Nutrition run
2019-11	–	First run on P. astreoides adult homogenates