Amplite™ Fluorimetric Peroxynitrite Quantification Kit *Green Fluorescence*

Image Viewer
Analyze with Quest Graph™Plan a serial dilution
Close (X)
1e+49.0e+36.0e+33.0e+3101- Dose-responseData legend Generated with Quest Graph™ Peroxynitrite (uM) RFU Hover mouse to interact
Peroxynitrite was measured with Amplite™ Fluorimetric Peroxynitrite Quantification Kit on a solid black 96-well plate using a Gemini microplate reader (Molecular Devices).
Loading...
 
Unit Size: Cat No: Price (USD): Qty:
100 Tests 16316 $345


Export item/cart as Excel file

Send item/cart as email
EXPORT TO EXCEL X

Export:
EXPORT TO EMAIL X
Important: We request your email address to ensure that the recipient(s) knows you intended for them to see the email, and that it is not junk mail.
Export:
Your Name*:
Your Email*:
Recipient Email*:
Your Personal Message:
Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

PlatformsFluorescence microplate reader
Storage Freeze (<-15 °C)
Minimize light exposure
Category Neurobiology
Reactive Oxygen Species
Related Cell Signaling
Secondary Reagents
Peroxynitrite (ONOO-) is a strong oxidizing species and a highly active nitrating agent. Peroxynitrite is formed from the reaction between superoxide radicals and nitric oxide generated in cells. It can damage a wide array of biomolecules including proteins, enzymes, lipids and nucleic acids, eventually contributing to cell death. Due to its extremely short half-life and low steady-state concentration, it has been challenging to detect and quantify peroxynitrite in solution. In order to address this need, AAT Bioquest's Amplite™ Fluorimetric Peroxynitrite Quantification Kit provides a sensitive tool to measure ONOO- in solution. DAX-J2™ PON Green 99 reacts with ONOO- to generate a bright green fluorescent product. It specifically reacts with ONOO- with high selectivity over other reactive oxygen species (ROS) and reactive nitrogen species (RNS). This kit can be used with a fluorescence microplate reader and spectrometer.




Protocol


Quick Preview

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

Important notes
Thaw all the kit components to room temperature before use. 

Key parameters
Instrument:Fluorescence microplate reader
Excitation:490 nm
Emission:530 nm
Cutoff:515 nm
Recommended plate:Solid black
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.

1. DAX-J2™ PON Green 99 stock solution (500X):
Add 20 µL of DMSO (Component C) into the vial of DAX-J2™ PON Green 99 (Component A) to make 500X stock solution. Note: 20 µL of reconstituted DAX-J2™ PON Green 99 stock solution is enough for 1 plate. Keep from light.

2. Peroxynitrite (ONOO¯ ) stock solution (not provided):
Peroxynitrite stock solution was synthesized according to literature report. Briefly, a mixture of sodium nitrite (0.6 M) and hydrogen peroxide (0.7 M) was acidified with hydrochloric acid (0.6 M), and sodium hydroxide (1.5 M) was added within 1 - 2 seconds to make the solution alkaline. The excess hydrogen peroxide was removed by passing the solution through a short column of manganese dioxide. The extinction coefficient of peroxynitrite solution in 0.1 M NaOH is 1670 M-1cm-1 at 302 nm. The ONOO¯ stock solution is not stable; we highly recommend make it fresh to use.

Preparation of standard solution
Peroxynitrite standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/16316

Dilute Peroxynitrite (ONOO¯) stock solution in Assay buffer (Component B) to have 20 µM ONOO¯ standard solution, and then perform 1:2 serial dilutions to get serially diluted ONOO¯ standard solution (O7 - O1).

Preparation of working solution

Add 20 μL of 500X DAX-J2™ PON Green 99 stock solution into 10 mL of Assay Buffer (Component B) and mix well. Note: This assay mixture is enough for one 96-well plate. Protect from light.

Sample experimental protocol

Table 1. Layout of ONOO¯ standards and test samples in a solid black 96-well microplate. O = ONOO¯ Standards (O1 - O7, 0.313 to 20 µM); BL = Blank Control; TS = Test Samples.

BL BL TS TS
O1 O1 ... ...
O2 O2 ... ...
O3 O3    
O4 O4    
O5 O5    
O6 O6    
O7 O7    

Table 2. Reagent composition for each well.

Well Volume Reagent
O1 - O7 50 µL Serial Dilutions (0.313 to 20 µM)
BL 50 µL assay buffer
TS 50 µL test sample
  1. Prepare ONOO¯ standards (O), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of working solution to each well of ONOO¯ standard, blank control, and test samples to make the total ONOO¯ assay volume of 100 µL/well. For a 384-well plate, add 25 µL of working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction at room temperature for 5 to 10 minutes, protected from light.

  4. Monitor the fluorescence increase at Ex/Em = 490/530 nm (cutoff at 515 nm) with a fluorescence plate reader.
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 Peroxynitrite samples. We recommend using the Online Four Parameter Logistics Calculator which can be found at:

https://www.aatbio.com/tools/four-parameter-logistic-4pl-curve-regression-online-calculator/

Figure 1. Peroxynitrite was measured with Amplite™ Fluorimetric Peroxynitrite Quantification Kit on a solid black 96-well plate using a Gemini 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

Branched-chain amino acids promote endothelial dysfunction through increased reactive oxygen species generation and inflammation
Authors: Olha Zhenyukh, Maria González-Amor, Raul R Rodrigues-Diez, Vanesa Esteban, Marta Ruiz-Ortega, Mercedes Salaices, Sebastian Mas, Ana M Briones, Jesus Egido
Journal: Journal of Cellular and Molecular Medicine (2018)

Fluorescent real-time quantitative measurements of intracellular peroxynitrite generation and inhibition
Authors: Zhen Luo, Qin Zhao, Jixiang Liu, Jinfang Liao, Ruogu Peng, Yunting Xi, Zhenjun Diwu
Journal: Analytical biochemistry (2017): 44--48

Gene silencing of endothelial von Willebrand Factor attenuates angiotensin II-induced endothelin-1 expression in porcine aortic endothelial cells
Authors: Anar Dushpanova, Silvia Agostini, Enrica Ciofini, Manuela Cabiati, Valentina Casieri, Marco Matteucci, Silvia Del Ry, Aldo Clerico, Sergio Berti, Vincenzo Lionetti
Journal: Scientific Reports (2016)






Documents

 
Safety Data Sheet (SDS)


Catalogs
1. Reactive Oxygen Species (ROS) Detection

Certificate of Analysis