Products

Total Antioxidant Capacity Assay Kit (FRAP)

K025 Catalogue No.
Overview
Product name
Total Antioxidant Capacity Assay Kit (FRAP)
Product code
K025
Storage Temperature
-20℃ for 12 months
Product Description
  • Oxidants, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), can generate free radicals that can cause severe oxidative damage to cellular lipids, membranes, proteins, and DNA. Antioxidants can scavenge these free radicals and prevent cellular oxidative stress by enzymatic and non-enzymatic mechanisms. Enzyme systems that function as antioxidants include catalase and peroxidase. Tocopherols, carotenes, vitamin A, and ubiquinols function as lipid-soluble antioxidants; whereas, glutathione and ascorbate are some of the water-soluble antioxidants. Measurement of the total non-enzymatic antioxidant capacity (TAC) of biological samples is indicative of their ability to counteract oxidative stress-induced damage in cells. TAC is used to provide insights into the development and treatment of oxidative-stress related disorders.
  • In the Total Antioxidant Capacity Assay Kit, either the concentration of the combination of both small molecule and protein antioxidants, or the concentration of only small molecule antioxidants can be determined. Under acidic condition, Fe3+-TPTZ is converted to Fe2+-TPTZ by both small molecules and proteins. The reduced Fe2+ion chelates with a colorimetric probe, giving a broad absorbance peak at 593 nm, which is proportional to the total antioxidant capacity. Because of acidic conditions, and the total plasma concentration of iron ion and ferrous ion in serum samples is usually lower than 10 μM, some endogenous interference factors can be inhibited. Things.
  • Antioxidant Fe3+-TPTZ ——————> Fe2+-TPTZ (blue)
Components


TPTZ Diluent

15 ml

-20°C

TPTZ solution

1.5 ml

-20°C, avoiding light

Detection buffer

1.5 ml

-20°C, avoiding light

FeSO4∙7H2O

1.5 g

-20°C, avoiding light

Positive Control (2mM)

0.1 ml

-20°C


Standard curve preparation
  • The FeSO4 solution should be freshly prepared for it is easy to be oxidized to ferric. If the color turn from light green to light yellow, discard it and prepare a new one .
  • Dissolve 13.9 mg FeSO4∙7H2O in 1 ml PBS, dilute the solution 10 times to make the concentration of 5 mM.
  • Prepare standard curve dilution as described in the table in 96 well plates.

concentration (mM)

FeSO4 solution (μl)

dH2O (μl)

1.5

6

14

1

4

16

0.75

3

17

0.5

2

18

0.25

1

19

0

0

20

Storage/Stability

The kit is shipped on ice pack and storage at -20°C for 12 months, avoiding light

Procedure

All samples and standards should be run in duplicate.

Sample Preparation
  1. Serum, plasma, saliva, urine, and culture media samples can be directly added to the wells. For plasma samples, it is suggested to use heparin or sodium citrate, not EDTA, for anticoagulation. According to the literature, the total antioxidant capacity of human serum or plasma was 0.5-2mM, saliva was 0.3-1mM, urine was 0.2-3mM.
  2. For cells or tissue samples, supernatant of prepared cells or tissue samples can be directly added to the wells. At the same time, protein concentration should be tested; final determination of total antioxidant capacity was usually expressed as the total antioxidant capacity per gram or microgram of protein, indicating a unit of mmol/mg or mmol/g.
  3. For other samples, Plant or Chinese herbal extract can be directly added to the wells, total antioxidant capacity can be expressed mmol/mg or mmol/g. If the concentration of antioxidant can be expressed as molar concentration, the total antioxidant capacity measured can be expressed by the relative total antioxidant capacity. For example, the measured absorbance of a 0.5mM antioxidant is the same as that of 1mM FeSO4, the relative total antioxidant capacity was 2.
Assay Reaction
  1. Mix the TPTZ Diluent, TPTZ solution, and Detection buffer with ratio of 10:1:1 to make the assay solution. Prepared for immediate use, operated on ice.
  2. Add 20 μl sample, positive control and deionized wate (blank control) to the wells.
  3. Add 180μl of assay solution to each well.
  4. Measure A593 (585-605nm can also) after incubation at 37°C for 3-5 minutes.