What is Amplex Red assay?
The Amplex® Red assay is based on the oxidation of 10-acetyl-3,7- dihydroxypenoxazine, which is catalyzed by horseradish peroxidase (HRP) in the presence of H2O2 to produce a red fluorescent oxidation product, resorufin, at a 1:1 ratio (Figure 1).
How does Amplex Red work?
Amplex Red, in the presence of peroxidase enzyme, reacts with H2O2 in a 1:1 stiochiometry to produce resorufin, a red fluorescent compound (Figure 1) which has an absorption and fluorescence emission maxima of 563 nm and 587 nm respectively [2].
Why is horseradish peroxidase important?
Horseradish peroxidase (HRP) mediates efficient conversion of many phenolic contaminants and thus has potential applications for pollution control. Such potentially important applications suffer however from the fact that the enzyme becomes quickly inactivated during phenol oxidation and polymerization.
What is h2o2 assay?
The Hydrogen Peroxide Assay is for the quantitative determination of hydrogen peroxide in colorless tissue culture media and buffers. For research use only. DESCRIPTION. Hydrogen Peroxide (H202) is a reactive oxygen metabolic by-product that serves as a key regulator for a number of oxidative stress-related states.
What is horseradish peroxidase HRP and what does it do?
Horseradish peroxidase (HRP) is an enzyme used to amplify signal in photometric assays by catalyzing the conversion of chromogenic or chemiluminescent substrates for the detection of targets such as proteins, carbohydrates, and nucleic acids.
What does 20 volume H2O2 solution mean?
20 volumes of H2O2 means 20 litres of oxygen is obtained at STP by the decomposition of 1 litre H2O2 solution 2H2O2→2H2O+O2 2 mole 1 mole 68 g 22.4 litre at STP 22.4 litres oxygen is obtained from 68 g H2O2 ∴ 20 litres oxygen is obtained from = 6822.4×20=60.7gH2O2 ∴ 1000 ml of H2O2 solution contains 60.7 g H2O2 100 ml …
What is the concentration of 6% hydrogen peroxide?
6% H2O2 in water = 6 g H2O2 in 100 mL water = 60 g H2O2 in 1 liter water. MW H2O2 = 34 g/mol. Therefore the molarity of your 6% solution is 60 g/34 g/mol = 1.765 mol in 1 liter = 1.765 M.
Why does TMB turn blue?
TMB turns blue in presence of H2O2 and Peroxidase. Sometimes metals also act as peroxidase mimic. Just avoid any peroxide and peroxidase in proximity. You should consider preparing a fresh stock.
How do you make 20mm H2O2?
To prepare a 20 mM solution take 2.05 ml of your 30% solution and fill to 1 L with water (20 mM x 1000 ml / 9.79 M). If, however, the concentration of your stock solution is given as v/v = volume per volume, then Arvind is correct in calculating the molarity of the stock solution as 12.7 M.
Which is better Amplex ultrared or Amplex Red?
Consequently, assays using Amplex Red as the substrate result in an increase in fluorescence, not a decrease—an inherently superior method for enzymatic assays. Amplex UltraRed reagent—the second-generation Amplex substrate—exhibits increased resistance to oxidation and works better in lower pH environments.
When to use Amplex Red reagent in peroxidase assay?
Furthermore, Amplex® Red reagent can be used as an ultrasensitive assay for peroxidase activity when H 2 O 2 is in excess. In the presence of peroxidase, the Amplex® Red reagent reacts with H 2 O 2 in a 1:1 stoichiometry to produce the red-fluorescent oxidation product, resorufin.
How is HRP used in Amplex Red reagent?
In the scopoletin assay, HRP catalyzes conversion of the fluorescent scopoletin to a nonfluorescent product. Unlike scopoletin, the Amplex Red reagent is a fluorogenic substrate with very low background fluorescence.
Which is better Amplex Red or scopoletin reagent?
Unlike scopoletin, the Amplex® Red reagent is a fluorogenic substrate with very low background fluorescence. Consequently, assays using Amplex® Red as the substrate result in an increase in fluorescence, not a decrease—an inherently superior method for enzymatic assays.