Amplite™ IR

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1.6e+31.2e+38004008642- Dose-ResponseData legend Generated with Quest Graph™ H2O2 (uM) RFU Hover mouse to interact
H2O2 dose response was measured in a solid black 96-well plate with Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit.
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1 mg 11009 $95


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Telephone: 1-800-990-8053
Fax: 1-408-733-1304
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Overview

Ex/Em (nm)646/667
MW~400
SolventDMSO
Storage Freeze (<-15 °C)
Minimize light exposure
Category Enzyme Detection
Horseradish Peroxidase (HRP)
Related
Our Amplite™ IR is a fluorogenic peroxidase substrate that generates near infrared fluorescence upon reaction with peroxidase and H2O2. It can be used to detect both H2O2 and peroxidase. Amplite™ IR generates a substance that has maximum absorption of 647 nm with maximum emission at 670 nm. This near infrared absorption and fluorescence minimize the assay background that is often caused by the autoabsorption and/or autofluorescence of biological samples that rarely absorb light beyond 600 nm. Unlike other HRP substrates such as dihydrofluoresceins and dihydrorhodamines, the air-oxidation of Amplite™ IR is minimal. Compared to Amplex Red™, Amplite™ IR generates the fluorescence that is pH-independent from pH 4 to 10. In addition, it has excellent water solubility. It is a superior alternative to Amplex Red™ for the detections that require low pH where Amplex Red™ has significantly reduced fluorescence. We have used Amplite™ IR to detect HRP in quite a few immunoassays. Amplite™ IR can also be used to detect trace amount of H2O2. Because H2O2 is produced in many enzymatic redox reactions, Amplite™ IR 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.




Calculators
Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Amplite™ IR to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.



Molarity calculator

Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
/ = x =
 






Spectrum Advanced Spectrum Viewer

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Move mouse over grid to display wavelength & intensity values.

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Wavelength (nm)





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

  1. Prepare 100 µM AmpliteTM IR with 0.8 U/mL peroxidase in phosphate buffer and add 50 µL in a well
  2. Add H2O2 standards or test samples (50 µL)
  3. Incubate at RT for 0-30 minutes
  4. Monitor fluorescence intensity at Ex/Em = 640/680 nm

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

Key parameters
Instrument:Fluorescence microplate reader
Excitation:640 nm
Emission:680 nm
Cutoff:650 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 IR stock solution:
Add appropriate amount of anhydrous DMSO to make 10 to 25 mM AmpliteTM IR stock solution.

Preparation of working solution

AmpliteTM IR working solution(2X):
In order to achieve final concentration per well of 50 to 100 µM in 50 mM phosphate buffer or buffer of your choice, make 100 to 200 µM concentration solution in a tube. 50 µL is required per well. Note: AmpliteTM IR 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. Note: We recommend using fresh stock solution every time you perform experiments.

Sample experimental protocol

Run H2O2 assay in supernatants

  1. Add 50 µL of 2X AmpliteTM IR working solution (from Step 1.2) 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 2X AmpliteTM IR working solution into each well.

  2. Incubate the reaction at room temperature for 0 to 30 minutes, protected from light.

  3. Monitor the fluorescence increase at Ex/Em = 640/680 nm with a fluorescence plate reader. Note: Amplite™ IR peroxidase substrate is easy to be self-oxidized, so read the fluorescence as soon as the H2O2 reaction mixture is added to increase the signal to noise ratio.

  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

 Run H2O2 assay for cells:

  1. Amplite™ IR can be used to measure the release of H2O2 from cells. The following is a suggested protocol that can be modified for your specific research needs.The AmpliteTM IR working solution should be prepared as Step 1.2 except that the phosphate buffer should be replaced with the media that is used in the cell culture system. Suggested media including (a) Krebs Ringers Phosphate Buffer (KRPB); (b). Hanks Balanced Salt Solution (HBSS); or (c) Serum-free media.

  2. Prepare cells in a 96-well plate (50-100 µL/well), and activate the cells as desired. Note: The negative controls (media alone and non-activated cells) are included for measuring background fluorescence.

  3. Add 50 µL of H2O2 reaction mixture to each well of the cells, and those of H2O2.Note: For a 384-well plate, add 25 µL of cells and 25 µL of H2O2 reaction mixture into each well.

  4. Incubate the reaction for 0 to 30 minutes at room temperature, protected from light.

  5. Monitor the fluorescence increase at Ex/Em = 640/ 680 nm with a fluorescence plate reader. Note: The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 670 nm. The absorption detection has lower sensitivity compared to fluorescence reading. Note: The fluorescence background increases with time, thus it is important to subtract the fluorescence intensity value of the blank wells for each data point.
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 H2O2 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. H2O2 dose response was measured in a solid black 96-well plate with Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit.

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