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Amplite™ IR

H2O2 dose response was measured in a solid black 96-well plate with Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit.
H2O2 dose response was measured in a solid black 96-well plate with Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit.
Ordering information
Price ()
Catalog Number11009
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
Molecular weight400
SolventDMSO
Spectral properties
Excitation (nm)648
Emission (nm)668
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

OverviewpdfSDSpdfProtocol


Molecular weight
400
Excitation (nm)
648
Emission (nm)
668
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.

Platform


Fluorescence microplate reader

Excitation640 nm
Emission680 nm
Cutoff650 nm
Recommended plateSolid black

Example protocol


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.

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.

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.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM250 µL1.25 mL2.5 mL12.5 mL25 mL
5 mM50 µL250 µL500 µL2.5 mL5 mL
10 mM25 µL125 µL250 µL1.25 mL2.5 mL

Molarity calculator

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

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Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)648
Emission (nm)668

Citations


View all 11 citations: Citation Explorer
Wearable materials with embedded synthetic biology sensors for biomolecule detection
Authors: Nguyen, Peter Q and Soenksen, Luis R and Donghia, Nina M and Angenent-Mari, Nicolaas M and de Puig, Helena and Huang, Ally and Lee, Rose and Slomovic, Shimyn and Galbersanini, Tommaso and Lansberry, Geoffrey and others,
Journal: Nature Biotechnology (2021): 1366--1374
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

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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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