Putnam Lab Total Antioxidant Capacity Protocol

Draft Protocol

Goal: To determine the total antioxidant capacity in adult coral tissue homogenate samples.

Protocol written for Montipora capitata and Pocillopora acuta adult coral samples from the Holobiont Integration 2018 project. Modified from Cell BioLabs OxiSelect Total Antioxidant Capacity (TAC) Assay Kit.

Materials Not Supplied in Kit

• Standard 96-well microtiter plates for use in microplate reader.
• 1N NaOH, 1X PBS, and DI water
• Methanol or other organic solvent for lipid-based samples
• Sonicator or homogenizer for samples preparations
• 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
• 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
• Spectrophotometer microplate reader capable of reading 490 nm

Reagent Preparation

1. 1X Reaction Buffer: Dilute the Reaction Buffer 1:100 with 1X PBS (hydrophilic) or with methanol (lipophilic). Mix to homogenity. Store the 1X Reaction Buffer at 4°C for up to 3 months.
2. 1X Copper Ion Reagent: Dilute the Copper Ion Reagent 1:100 with deionized water (hydrophilic) or with methanol (lipophilic). Mix to homogenity. Store the 1X Copper Ion Reagent at 4°C for up to 3 months.
3. 1X Stop Solution: Dilute the Stop Solution 1:10 with deionized water (hydrophilic) or with methanol (lipophilic). Mix to homogenity. Store the 1X Stop Solution at 4°C for up to three months.

Sample Preparation

1. Sonicate or homogenize tissue sample on cold PBS. For Holobiont Integration project, this step is done during the airbrushing protocol. The following steps are done with the Total Protein/Total Antioxidant Capacity aliquot.
2. Centrifuge at 10,000 xg for 10 minutes at 4°C.
3. Aliquot the supernatant for storage at -80°C, protein determination, and subsequent TAC assay. Use this supernatant for the total protein protocol.

Uric Acid Standard Curve Preparation

1. Add 10 mg of the Uric Acid powder to 1 mL of 1N NaOH (10 mg/mL ratio) to create a 60 nM Uric Acid standard.
2. Add 100 μL of the 60 mM Uric Acid standard to 2.9 mL of DI water to create a 2 mM solution of Uric Acid.
3. Prepare a series of the remaining Uric Acid standards according to the table below. Prepare in DI water.

Assay Protocol Each Uric Acid Standard and sample should be assayed in duplicate for replication. A freshly prepared standard curve should be used each time the assay is performed.

1. Using a pipette, mix the sample in the microcentrifuge tube. Add 20 μL of the diluted Uric Acid Standards or samples to the 96-well microtiter plate.
2. Add 180 μL of the 1X Reaction Buffer to each well.
3. Measure an initial absorbance at 490 nm.
4. Add 50 μL of the 1X Copper Ion Reagent into each well.
5. Incubate 5 minutes on an orbital shaker.
6. Add 50 μL of 1X Stop Solution to each well. This terminates the reaction.
7. Measure the absorbance for each well at 490 nm.