Ascorbic acid (the L-enantiomer commonly known as vitamin C) is an important antioxidant found in living organisms and applied as additives in food and other industrial processes. By reacting with reactive oxygen species, it protects the cell from oxidative damages. BioAssay Systems' method provides a simple, direct and high-throughput assay for measuring ascorbic acid. In this assay, ascorbic acid is oxidized by ascorbate oxidase resulting in the production of H2O2 which reacts with a specific dye to form a pink colored product. The color intensity at 570nm or fluorescence intensity (530/585 nm) is directly proportional to the ascorbic acid concentration in the sample.

Use 20 μL samples. Linear detection range: colorimetric assay 6 to 1000 μM, fluorimetric assay 1 to 100 μM ascorbic acid.

APPLICATIONS

Assays: ascorbic acid in biological samples such as serum, plasma, urine, saliva, milk, tissue, and cell culture.

Drug Discovery/Pharmacology: effects of drugs on ascorbic acid metabolism.

KIT CONTENTS

Assay Buffer: 10 mL Enzyme Mix: 120 μL

Dye Reagent: 120 μL Standard: 400 μL 10 mM ascorbic acid

Storage conditions. The kit is shipped on ice. Store all components at -20°C. Shelf life of three 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.

Note: SH-containing reagents (e.g. b–mercaptoethanol, dithiothreitol, > 5 μM) are known to interfere in this assay and should be avoided in sample preparation.

Sample treatment: liquid samples such as serum and plasma can be assayed directly. Tissue and cell (106-107) lysates can be prepared by homogenization in cold 1 x PBS and centrifugation (5 min at 14,000 rpm). Use clear supernatants for assay. Milk samples should be cleared by mixing 600 μL milk with 100 μL 6 N HCl. Centrifuge 5 min at 14,000 rpm. Transfer 300 μL supernatant into a clean tube and neutralize with 50 μL 6 N NaOH. The neutralized supernatant is ready for assay (dilution factor n = 1.36).

1. Equilibrate all components to room temperature. Briefly centrifuge the tubes before opening. Keep thawed tubes on ice during assay.

2. Standards: mix 22 μL 10 mM Standard with 198 μL dH2O (final 1000 μM). Dilute standard in dH2O as follows..

RSAMPLE and RBLANK are optical density or fluorescence intensity readings of the Sample and H2O Blank, respectively. n is the sample dilution factor.

Conversions: 1 mM ascorbic acid equals 17.6 mg/dL, 0.0176% or 176 ppm.

Pipetting devices, centrifuge tubes, clear flat-bottom uncoated 96-well plates, optical density plate reader; black flat-bottom uncoated 96-well plates, fluorescence plate reader.