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Amplite® Red

HRP dose response was measured with Amplite® Fluorimetric Peroxidase Assay Kit in a black plate using a Gemini fluorescence microplate reader (Molecular Devices).
HRP dose response was measured with Amplite® Fluorimetric Peroxidase Assay Kit in a black plate using a Gemini fluorescence microplate reader (Molecular Devices).
Ordering information
Price ()
Catalog Number11011
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
SolventDMSO
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
StorageFreeze (< -15 °C); Desiccated; Minimize light exposure
UNSPSC12171501
Alternative formats
Amplite® Blue

OverviewpdfSDSpdfProtocol


Excitation (nm)
571
Emission (nm)
584
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.

Platform


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff550 nm
Recommended plateSolid black

Example protocol


AT A GLANCE

Protocol summary for Peroxidase (HRP) with AmpliteTM Red (for one 96 well black plate)

  1. Prepare and add 1X AmpliteTM Red working solution with 200 mM 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 notes
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 AmpliteTM Red (for one 96 well black plate)

  1. Prepare 1X AmpliteTM 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 notes
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 SOLUTION

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.

AmpliteTM Red stock solution (250X):
Add 200 mL of anhydrous DMSO into the vial, mixed well. The stock solution should be used promptly. Any unused solution need to be aliquoted and refrozen at < -20 oNote: Avoid repeated freeze-thaw cycles, and protect from light.

PREPARATION OF WORKING SOLUTION

1. AmpliteTM Red Peroxidase working solution (1X):
Add 20 μL of AmpliteTM Red stock solution (250X) in 5 mL of 50 mM phosphate buffer or buffer of your choice, pH 7 with 200 mM H2O2Note: AmpliteTM 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.

2. AmpliteTM Red H2O2 working solution (1X):
Add 20 μL of AmpliteTM 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: AmpliteTM 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 AmpliteTM 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 AmpliteTM 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.

H2O2 assay in supernatants

  1. Add 50 µL of 1X AmpliteTM 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 AmpliteTM 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


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)571
Emission (nm)584

Product family


Name
Amplite® Blue

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 & 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
Ordered cleavage of myeloperoxidase ester bonds releases active site heme leading to inactivation of myeloperoxidase by benzoic acid hydrazide analogs
Authors: Huang, Jiansheng and Smith, Forrest and Panizzi, Peter
Journal: Archives of biochemistry and biophysics (2014): 74--85
Raising the shields: PCR in the presence of metallic surfaces protected by tailor-made coatings
Authors: Scherag, Frank D and Br, undefined and stetter, Thomas and Rühe, Jürgen
Journal: Colloids and Surfaces B: Biointerfaces (2014): 576--582
Measuring myeloperoxidase activity in biological samples
Authors: Pulli, Benjamin and Ali, Muhammad and Forghani, Reza and Schob, Stefan and Hsieh, Kevin LC and Wojtkiewicz, Gregory and Linnoila, Jenny J and Chen, John W
Journal: PLoS One (2013): e67976
Micro-volume wall-less immunoassays using patterned planar plates
Authors: Kozak, Katherine R and Wang, Jianyong and Lye, Melvin and Takkar, Rashi and Kim, Namyong and Lee, Hyunjae and Jeon, Noo Li and Lin, Kedan and Zhang, Crystal and Wong, Wai Lee T and others, undefined
Journal: Lab on a Chip (2013): 1342--1350
Distinguishing inflammation from tumor and peritumoral edema by myeloperoxidase magnetic resonance imaging
Authors: Kleijn, Anne and Chen, John W and Buhrman, Jason S and Wojtkiewicz, Gregory R and Iwamoto, Yoshiko and Lamfers, Martine L and Stemmer-Rachamimov, Anat O and Rabkin, Samuel D and Weissleder, Ralph and Martuza, Robert L and others, undefined
Journal: Clinical Cancer Research (2011): 4484--4493

References


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A new, sensitive method for enzyme kinetic studies of scarce glucosides
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K+-independent actions of diazoxide question the role of inner membrane KATP channels in mitochondrial cytoprotective signaling
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A Role for Mitochondrial Dysfunction in Perpetuating Radiation-Induced Genomic Instability
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