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Amplite® Red

Our Amplite® Red is a sensitive fluorogenic peroxidase substrate that generates a highly red fluorescent product that has maximum absorption of 571 nm and maximum emission of 585 nm. Unlike other HRP substrates such as dihydrofluoresceins and dihydrorhodamines, the air-oxidation of Amplite® Red is minimal. Amplite® Red is one of the most sensitive and stable fluorogenic probes for detecting HRP and H2O2. Amplite® Red has been widely used to detect HRP in many immunoassays. On the other hand, Amplite® Red can also be used to detect trace amount of H2O2. The Amplite® Red-based H2O2 detection is at least one order of magnitude more sensitive than the commonly used scopoletin assay for H2O2. Because H2O2 is produced in many enzymatic redox reactions, Amplite® Red can be used in coupled enzymatic reactions to detect the activity of many oxidases and/or related enzymes/substrates or cofactors such as glucose, acetylcholine and cholesterol, L-glutamate, amino acids, etc.

Example protocol

AT A GLANCE

Protocol summary for Peroxidase (HRP) with Amplite® Red (for one 96 well black plate)
  1. Prepare and add 1X Amplite® Red working solution with 200 µM H2O2 in phosphate buffer (50 µL)

  2. Add Peroxidase standards or test samples (50 µL)
  3. Incubate at room temperature for 10-30 minutes

  4. Monitor fluorescence intensity at Ex/Em = 540/590 nm
Important Note

The following is the recommended protocol for peroxidase assay in solution. The protocol only provides a guideline, should be modified according to the specific needs.

Thaw one of each kit component at room temperature before starting the experiment.

Protocol Summary for H2O2 with Amplite® Red (for one 96 well black plate)
  1. Prepare 1X Amplite® Red H2O2 working solution with 0.4 U/mL peroxidase in phosphate buffer (50 µL)

  2. Add Peroxidase standards or test samples (50 µL)
  3. Incubate at room temperature for 10-30 minutes
  4. Monitor fluorescence intensity at Ex/Em = 540/590 nm
Important Note

The following is the recommended protocol for H2O2 assay in solution. The protocol only provides a guideline, should be modified according to the specific needs.

Thaw one of each kit component at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Amplite® Red stock solution (250X)

Add 200 µL of anhydrous DMSO into the vial and mix well. The stock solution should be used promptly. Any unused solution needs to be aliquoted and refrozen at <-20°C.

Note: Avoid repeated freeze-thaw cycles and protect from light.

PREPARATION OF WORKING SOLUTION

Amplite® Red Peroxidase working solution (1X)

Add 20 μL of Amplite® Red stock solution (250X) in 5 mL of 50 mM phosphate buffer or buffer of your choice, pH 7, with 200 μM H2O2.

Note: Amplite® Red is unstable in the presence of thiols such as DTT and b-mercaptoethanol. Thiols higher than 10 μM (final concentration) could significantly decrease the assay dynamic range. NADH and glutathione (reduced from GSH) may interfere with the assay.

Amplite® Red H2O2 working solution (1X)

Add 20 μL of Amplite® Red stock solution (250X) in 5 mL of 50 mM phosphate buffer or buffer of your choice, pH 7 with 0.4 units/mL peroxidase.

Note: Amplite® Red is unstable in the presence of thiols such as DTT and b-mercaptoethanol. Thiols higher than 10 μM (final concentration) could significantly decrease the assay dynamic range. NADH and glutathione (reduced from GSH) may interfere with the assay.

SAMPLE EXPERIMENTAL PROTOCOL

Peroxidase assay in supernatants
  1. Add 50 µL of 1X Amplite® Red peroxidase working solution into each well of the peroxidase standard, blank control, and test samples to make the total peroxidase assay volume of 100 µL/well.

    Note: For a 384-well plate, add 25 µL of sample and 25 µL of 1X Amplite® Red peroxidase working solution into each well.

  2. Incubate the reaction at room temperature for 10 to 30 minutes, protected from light.
  3. Monitor the fluorescence increase at Ex/Em = 540/590 nm with a fluorescence plate reader.
  4. The fluorescence in blank wells (with the assay buffer only) is used as a control, and is subtracted from the values for those wells with the peroxidase reactions.
H202 assay in supernatants
  1. Add 50 µL of 1X Amplite® Red H2O2 working solution into each well of the H2O2 standard, blank control, and test samples to make the total H2O2 assay volume of 100 µL/well.

    Note: For a 384-well plate, add 25 µL of sample and 25 µL of 1X Amplite® Red H2O2 working solution into each well.

  2. Incubate the reaction at room temperature for 10 to 30 minutes, protected from light.
  3. Monitor the fluorescence increase at Ex/Em = 540/590 nm with a fluorescence plate reader.
  4. The fluorescence in blank wells (with the assay buffer only) is used as a control, and is subtracted from the values for those wells with the H2O2 reactions.

Spectrum

Citations

View all 10 citations: Citation Explorer
Patterned Photonic Nitrocellulose for Pseudo-Paper ELISA
Authors: Chi, Junjie and Gao, Bingbing and Sun, Mi and Zhang, Fengling and Su, Enben and Liu, Hong and Gu, Zhongze
Journal: Analytical Chemistry (2017)
Spinal Cord Inflammation: Molecular Imaging after Thoracic Aortic Ischemia Reperfusion Injury
Authors: Albadawi, Hassan and Chen, John W and Oklu, Rahmi and Wu, Yue and Wojtkiewicz, Gregory and Pulli, Benjamin and Milner, John D and Cambria, Richard P and Watkins, Michael T
Journal: Radiology (2016): 152222
Myeloperoxidase--Hepatocyte--Stellate Cell Cross Talk Promotes Hepatocyte Injury and Fibrosis in Experimental Nonalcoholic Steatohepatitis
Authors: Pulli, Benjamin and Ali, Muhammad and Iwamoto, Yoshiko and Zeller, Matthias WG and Schob, Stefan and Linnoila, Jenny J and Chen, John W
Journal: Antioxidants &amp; redox signaling (2015): 1255--1269
Myeloperoxidase Nuclear Imaging for Epileptogenesis
Authors: Zhang, Yinian and Seeburg, Daniel P and Pulli, Benjamin and Wojtkiewicz, Gregory R and Bure, Lionel and Atkinson, Wendy and Schob, Stefan and Iwamoto, Yoshiko and Ali, Muhammad and Zhang, Wei and others, undefined
Journal: Radiology (2015): 822--830

References

View all 61 references: Citation Explorer
K+-independent actions of diazoxide question the role of inner membrane KATP channels in mitochondrial cytoprotective signaling
Authors: Drose S, Br and t U, Hanley PJ.
Journal: J Biol Chem (2006): 23733
Real-time assessment of spatial and temporal coupled catalysis within polyelectrolyte microcapsules containing coimmobilized glucose oxidase and peroxidase
Authors: Stein EW, Volodkin DV, McShane MJ, Sukhorukov GB.
Journal: Biomacromolecules (2006): 710
O2 Delivery and Redox State are Determinants of Compartment-specific Reactive Oxygen Species in Myocardial Reperfusion
Authors: Stoner JD, Clanton TL, Aune SE, Angelos MG.
Journal: Am J Physiol Heart Circ Physiol. (2006)
Fibrillar beta-amyloid peptide Abeta1-40 activates microglial proliferation via stimulating TNF-alpha release and H2O2 derived from NADPH oxidase: a cell culture study
Authors: Jekabsone A, M and er PK, Tickler A, Sharpe M, Brown GC.
Journal: J Neuroinflammation (2006): 24
Biochemical activity of reactive oxygen species scavengers do not predict retinal ganglion cell survival
Authors: Schlieve CR, Lieven CJ, Levin LA.
Journal: Invest Ophthalmol Vis Sci (2006): 3878
Page updated on October 11, 2024

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Catalog Number11011
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Physical properties

Molecular weight

N/A

Solvent

DMSO

Spectral properties

Excitation (nm)

571

Emission (nm)

584

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure; Desiccated
UNSPSC12171501

Platform

Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff550 nm
Recommended plateSolid black