Amplite® Red
Overview | ![]() ![]() |
Molecular weight N/A | Excitation (nm) 571 | Emission (nm) 584 |
Platform
Fluorescence microplate reader
Excitation | 540 nm |
Emission | 590 nm |
Cutoff | 550 nm |
Recommended plate | Solid black |
Example protocol
AT A GLANCE
Protocol summary for Peroxidase (HRP) with AmpliteTM Red (for one 96 well black plate)
- Prepare and add 1X AmpliteTM Red working solution with 200 mM H2O2 in phosphate buffer (50 µL)
- Add Peroxidase standards or test samples (50 µL)
- Incubate at room temperature for 10-30 minutes
- 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)
- Prepare 1X AmpliteTM Red H2O2 working solution with 0.4 U/mL peroxidase in phosphate buffer (50 µL)
- Add Peroxidase standards or test samples (50 µL)
- Incubate at room temperature for 10-30 minutes
- 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
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 o. Note: 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 H2O2. 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.
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
- 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.
- Incubate the reaction at room temperature for 10 to 30 minutes, protected from light.
- Monitor the fluorescence increase at Ex/Em = 540/590 nm with a fluorescence plate reader.
- 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
- 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.
- Incubate the reaction at room temperature for 10 to 30 minutes, protected from light.
- Monitor the fluorescence increase at Ex/Em = 540/590 nm with a fluorescence plate reader.
- 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.
Citations
Authors: Milton, Amber
Journal: (2017)
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)
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
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
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
Authors: Huang, Jiansheng and Smith, Forrest and Panizzi, Peter
Journal: Archives of biochemistry and biophysics (2014): 74--85
Authors: Scherag, Frank D and Br, undefined and stetter, Thomas and Rühe, Jürgen
Journal: Colloids and Surfaces B: Biointerfaces (2014): 576--582
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
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
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
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References
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Application notes
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