Amplite™ Red

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1e+49.0e+36.0e+33.0e+33.22.41.60.8- Dose-ResponseData legend Generated with Quest Graph™ HRP (mU/mL) RFU Hover mouse to interact
HRP dose response was measured with Amplite™ Fluorimetric Peroxidase Assay Kit in a black plate using a Gemini fluorescence microplate reader (Molecular Devices).
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1000 Assays 11011 $95


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Overview

Ex/Em (nm)570/583
MWN/A
SolventDMSO
Storage Freeze (<-15 °C)
Minimize light exposure
Category Enzyme Detection
Horseradish Peroxidase (HRP)
Related
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.




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Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
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.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:540 nm
Emission:590 nm
Cutoff:550 nm
Recommended plate:Solid black
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.
Example data analysis and figures

The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate HRP samples. We recommend using the Online Linear Regression Calculator which can be found at:

https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator

Figure 1. HRP dose response was measured with Amplite™ Fluorimetric Peroxidase Assay Kit in a black plate using a Gemini fluorescence microplate reader (Molecular Devices).

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References

Assessment of Tofacitinib and Ruxolitinib and their Anti Inflammatory Effects on Myeloperoxidase
Authors: Amber Milton
Journal: (2017)

Patterned Photonic Nitrocellulose for Pseudo-Paper ELISA
Authors: Junjie Chi, Bingbing Gao, Mi Sun, Fengling Zhang, Enben Su, Hong Liu, Zhongze Gu
Journal: Analytical Chemistry (2017)

Spinal Cord Inflammation: Molecular Imaging after Thoracic Aortic Ischemia Reperfusion Injury
Authors: Hassan Albadawi, John W Chen, Rahmi Oklu, Yue Wu, Gregory Wojtkiewicz, Benjamin Pulli, John D Milner, Richard P Cambria, Michael T Watkins
Journal: Radiology (2016): 152222

Myeloperoxidase Nuclear Imaging for Epileptogenesis
Authors: Yinian Zhang, Daniel P Seeburg, Benjamin Pulli, Gregory R Wojtkiewicz, Lionel Bure, Wendy Atkinson, Stefan Schob, Yoshiko Iwamoto, Muhammad Ali, Wei Zhang
Journal: Radiology (2015): 822--830

Myeloperoxidase--Hepatocyte--Stellate Cell Cross Talk Promotes Hepatocyte Injury and Fibrosis in Experimental Nonalcoholic Steatohepatitis
Authors: Benjamin Pulli, Muhammad Ali, Yoshiko Iwamoto, Matthias WG Zeller, Stefan Schob, Jenny J Linnoila, John W Chen
Journal: Antioxidants & redox signaling (2015): 1255--1269

Ordered cleavage of myeloperoxidase ester bonds releases active site heme leading to inactivation of myeloperoxidase by benzoic acid hydrazide analogs
Authors: Jiansheng Huang, Forrest Smith, Peter Panizzi
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: Frank D Scherag, Thomas Brandstetter, Jürgen Rühe
Journal: Colloids and Surfaces B: Biointerfaces (2014): 576--582

Measuring myeloperoxidase activity in biological samples
Authors: Benjamin Pulli, Muhammad Ali, Reza Forghani, Stefan Schob, Kevin LC Hsieh, Gregory Wojtkiewicz, Jenny J Linnoila, John W Chen
Journal: PLoS One (2013): e67976

Micro-volume wall-less immunoassays using patterned planar plates
Authors: Katherine R Kozak, Jianyong Wang, Melvin Lye, Rashi Takkar, Namyong Kim, Hyunjae Lee, Noo Li Jeon, Kedan Lin, Crystal Zhang, Wai Lee T Wong
Journal: Lab on a Chip (2013): 1342--1350

Distinguishing inflammation from tumor and peritumoral edema by myeloperoxidase magnetic resonance imaging
Authors: Anne Kleijn, John W Chen, Jason S Buhrman, Gregory R Wojtkiewicz, Yoshiko Iwamoto, Martine L Lamfers, Anat O Stemmer-Rachamimov, Samuel D Rabkin, Ralph Weissleder, Robert L Martuza
Journal: Clinical Cancer Research (2011): 4484--4493






Resources
 
Safety Data Sheet (SDS)


Documents
1. Enzyme Probes & Assay Kits

Certificate of Analysis