What is Amplex UltraRed?

Recommendations. Recommendations. Our Amplex UltraRed reagent improves upon the performance of our unique Amplex Red reagent, offering brighter fluorescence and enhanced sensitivity on a per-mole basis in peroxidase or peroxidase-coupled enzyme assays.

What is the 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 is a substrate for use with HRP containing assays [2]. 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].

What is a horseradish peroxidase assay?

Introduction. Horseradish peroxidase (HRP) is an enzyme that when in the presence of hydrogen peroxide, oxidizes a substrate (oxygen donor), resulting in a color change that is quantifiable. There are about 40 HRP isoenzymes.

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.

Why is HRP used in IHC?

Antibody–HRP conjugates are superior to antibody–AP conjugates with respect to the specific activities of both the enzyme and antibody. In addition, its high turnover rate, good stability, low cost and wide availability of substrates makes HRP the enzyme of choice for most IHC applications.

What is the purpose of horseradish peroxidase?

Horseradish peroxidase (HRP) has the ability to catalyze the transfer of two electrons from a substrate to hydrogen peroxide to generate water and an oxidized substrate. HRP is frequently used in conjugates to detect the presence of a protein target.

How do you test the purity of hydrogen peroxide?

Pipette 50.0 mL (full pipette) of the hydrogen peroxide stock solution of approx. 1000 mg/Linto a 500-mL conical flask, dilute with 200 mL of distilled water, and add 30 mL of sulfuric acid 25 %. Titrate with a 0.02 mol/L potassium permanganate solution until the color changes to pink.

What color does HRP produce?

Colorimetric substrates for ELISA applications

Substrate	Enzyme	Absorbance and color
OPD Substrate	HRP	490 nm (450 nm) Green (Orange)
PNPP (p-Nitrophenyl Phosphate)
1-Step PNPP Substrate Solution	AP	405 nm Yellow
PNPP Substrate	AP	405 nm Yellow

What is TMB substrate?

TMB (3,3′,5,5′-Tetramethylbenzidine) is a substrate for horseradish peroxidase (HRP), a common label conjugated to antibodies for applications such as immunohistochemistry, western blotting, and ELISA. The combination of HRP and hydrogen peroxide causes the oxidation of TMB, resulting in the formation of a blue color.

What is HRP and TMB ELISA?

Description. 3,3’5,5′-Tetramethylbenzidine (TMB) is a safe-sensitive substrate used for the detection of Horseradish Peroxidase (HRP) activity. It yields a blue colour (Amax = 652nm) and changes to yellow (Amax = 450nm) upon the addition of either sulfuric or phosphoric acid.

What is the role of hydrogen peroxide in the assay?

The Hydrogen Peroxide Assay is for the quantitative determination of hydrogen peroxide in colorless tissue culture media and buffers. For research use only. 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 the pH level of hydrogen peroxide?

Hydrogen peroxide is acid as it has a pH of 6.2. However, the pH value depends upon the concentration as well as the temperature of the solution.

What is the principle of hydrogen peroxide assay?

It uses a color reagent that contains xylenol orange dye in an acidic solution with sorbitol and ammonium iron sulfate that reacts to produce a purple color in proportion to the concentration of H2O2 in the sample being tested.

How do you calculate assay hydrogen peroxide?

Weigh a 50-mL beaker to the nearest 0.1 mg. 2. Using a Mohr pipette, add to the 50-mL beaker: • ± 5 g of sample for 20% to 35% H2O2 • ± 3.5 g of sample for 50% H2O2 • ± 2.5 g of sample for 70% H2O2 • a proportionally larger sample for residual H2O2 (e.g., about 20 g for 1% H2O2).

How does HRP turn TMB blue?

TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP, and the addition of an appropriate stop solution gives a clear yellow color that absorbs at 450nm.

What are Amplex Red and Amplex ultrared coupled assays?

Amplex Red– and Amplex UltraRed–coupled assays permit the ultrasensitive quantitation of a diverse assortment of analytes, including glucose, galactose, cholesterol, glutamic acid, xanthine (or hypoxanthine), uric acid, choline and acetylcholine, inorganic phosphate and pyrophosphate, as well as hydrogen peroxide.

What is the Amplex Red uric acid/uricase assay kit?

The Amplex® Red Uric Acid/Uricase Assay Kit provides a sensitive and simple method for detecting uric acid or uricase using either a fluorescence microplate reader or fluorometer.

How do you store Amplex ultrared reagent?

The Amplex UltraRed reagent is also less sensitive to pH, and exhibits improved stability in the presence of hydrogen peroxide (H 2O2) or thiols such as dithiothreitol (DTT). For Research Use Only. Not for use in diagnostic procedures. Store in freezer (-5 to -30°C). Convenient, on-site access to the products you need. Learn more.

What is the range of acetylcholine levels in Amplex® Red assays?

By providing an excess of AChE in the assay, the kit can also be used to detect acetylcholine levels as low as 0.3 µM, with a range of detection from 0.3 µM to 100 µM acetylcholine. A wide variety of validated Amplex® Red assays are available for studying cell signaling and lipids, neurobiology, inflammation and immune function, and metabolism.