KEY FEATURES

Sensitive and accurate. Use 20 μL sample. Linear detection range from 1.5 to 1000 μM Trolox equivalents.

Simple and high-throughput. The procedure involves addition of a single working reagent and incubation for 10 min. Can be readily automated as a high-throughput assay for thousands of samples per day.

APPLICATIONS

Direct Assays: serum, plasma, urine, saliva and other biological samples, food and beverages.

Drug Discovery/Pharmacology: effects of drugs on TAC.

KIT CONTENTS

Reagent A: 12 mL Reagent B: 1 mL

Standard: 100 μL 50 mM Trolox

Storage conditions. The kit is shipped at room temperature. Store Reagent A at room temperature and other components at -20 °C. Shelf life of six months after receipt.

Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

Samples should not contain any metal chelators (e.g. EDTA) and should be clear and free of any turbidity or particles. Liquid samples (e.g. nonhemolyzed serum, plasma) can be assayed directly. Cell lysate is prepared by homogenizing or sonicating cells in ice-cold 1 x PBS and centrifugation for 10 min at 14,000 rpm to pellet any debris. Use the clear supernatant for the assay. If not assayed immediately, freeze supernatant at -80°C (stable for 1 month).

1. Standards and Samples. Equilibrate all components to room temperature. Briefly centrifuge Reagent B and Standard before opening. Mix 5 μL of the standard with 245 μL dH2O (final 1 mM Trolox). Dilute standards as shown in the Table below. Transfer 20 μL standards into wells of a clear flat-bottom 96-well plate.

Transfer 20 μL of each sample into separate wells of the 96-well plate.

Note: for unknown samples, perform several dilutions to ensure that TAC is within the linear range of 1.5 to 1000 μM Trolox equivalents.

2. Assay. Prepare enough Working Reagent for Sample and Standard wells by mixing, for each assay well, 100 μL Reagent A and 8 μL Reagent B. Add 100 μL Working Reagent to all assay wells. Tap plate to mix. Incubate 10 min at room temperature.

3. Read OD570nm on a plate reader.

Note: if calculated TAC is higher than 1000 μM Trolox equivalents, dilute sample in dH2O and repeat assay. Multiply the results by the dilution factor.

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