Amplite® Fluorimetric Goat Anti-Mouse IgG-HRP Conjugate ELISA Assay Kit *Red Fluorescence*
|Shipping||Standard overnight for United States, inquire for international|
|H-phrase||H303, H313, H333|
|Intended use||Research Use Only (RUO)|
|R-phrase||R20, R21, R22|
Fluorescence microplate reader
|Recommended plate||Solid black|
AT A GLANCE
- Prepare ELISA plate
- Prepare peroxidase working solution
- Add 100 µL/well of peroxidase working solution into the ELISA plate
- Incubate at room temperature for 15 - 60 minutes
- Monitor fluorescence intensity at Ex/Em = 540/590 nm
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. Amplite™ Red Peroxidase Substrate stock solution (200X):
Add 250 µL of DMSO (Component D) into the vial of Amplite™ Red Peroxidase Substrate (Component A). Note: 50 µL of the Amplite™ Red peroxidase substrate stock solution is enough for 1 plate.
2. H2O2 stock solution (20 mM):
Add 22.7 µL of 3% H2O2 (0.88 M, Component B) into 977 µL of Assay Buffer (Component C). Note: The diluted H2O2 stock solution is not stable. The unused portion should be discarded.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11540
Mouse IgG (not included) can be used to generate a standard curve. Dilute total mouse IgG with 0.2 M sodium bicarbonate buffer (pH 9.4) into microplate, using an initial concentration of 3 ug/mL and 1:3 dilution factor.
PREPARATION OF WORKING SOLUTION
1. Goat anti-mouse IgG-HRP conjugate working solution:
Add 2 µL of goat anti-mouse IgG-HRP conjugate (Component E) to 10 mL of PBS with 1% BSA (PBS-BSA, not included). Note: 10 mL of goat anti-mouse IgG-HRP conjugate working solution is enough for 1 plate. The concentration of this goat anti-mouse IgG-HRP conjugate working solution is recommended as an initial concentration to try. The optimal concentration for each particular application should be determined empirically.
2. Peroxidase working solution:
Add 50 μL of Amplite™ Red Peroxidase Substrate stock solution (200X) and 100 μL of H2O2 stock solution (20 mM) into 9.85 mL of Assay Buffer (Component C) to make a total volume of 10 mL Peroxidase working solution. Keep from light.
SAMPLE EXPERIMENTAL PROTOCOL
Prepare ELISA plate:
- Perform all necessary ELISA preparation steps.
- Wash the ELISA wells three times with PBS containing 0.02% to 0.05% Tween® 20 (PBS-Tween) and drain.
- Add 100 µL of prepared goat anti-mouse IgG-HRP conjugate working solution into each well.
- Incubate at room temperature for 30 minutes. Drain off the HRP conjugate.
- Wash the wells three times with PBS-Tween and drain.
Run peroxidase assay in ELISA plate:
- Add 100 µL of peroxidase working solution into each drained microplate well containing the samples and controls.
- Incubate the reaction at room temperature for 30 minutes or longer, protected from light.
- Monitor the fluorescence increase with a fluorescence plate reader at excitation 530 - 570 nm (optimal at 540 nm) and emission 590 - 600 nm. Note: The plate can also be read by an absorbance microplate reader at the wavelength of 576 ± 5 nm. However, the absorption detection has lower sensitivity compared to fluorescence reading.
|Name||Excitation (nm)||Emission (nm)|
|Amplite® Fluorimetric Goat Anti-Rabbit IgG-HRP Conjugate ELISA Assay Kit *Red Fluorescence*||571||584|
Authors: Yu, Wenbin and Ma, Bo and Zhao, Wei and Liu, Jingtao and Yu, Hao and Tian, Zhihua and Fan, Zhiqin and Han, Haibo
Journal: American Journal of Translational Research (2020): 5399
Authors: Milton, Amber
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: Rajashekhara E, Hosoda A, Sode K, Ikenaga H, Watanabe K.
Journal: Biotechnol Prog (2006): 334
Authors: Fortelius M, Mattjus P.
Journal: Chem Phys Lipids (2006): 103
Authors: Azuine MA, Patel SJ, Lakshman MR.
Journal: Neurochem Int (2006): 67
Authors: Morten KJ, Ackrell BA, Melov S.
Journal: J Biol Chem (2006): 3354
Authors: Mazura P, Fohlerova R, Brzobohaty B, Kiran NS, J and a L., undefined
Journal: J Biochem Biophys Methods (2006): 55
Authors: Rinaldi M, Moroni P, Paape MJ, Bannerman DD.
Journal: Vet Immunol Immunopathol. (2006)
Authors: Fruk L, Muller J, Niemeyer CM.
Journal: Chemistry (2006): 7448
Authors: Drose S, Br and t U, Hanley PJ.
Journal: J Biol Chem (2006): 23733
Authors: Kim GJ, Fiskum GM, Morgan WF.
Journal: Cancer Res (2006): 10377
Authors: Sullivan JC, Sasser JM, Pollock JS.
Journal: Am J Physiol Regul Integr Comp Physiol. (2006)
Acetylcholinesterase Inhibitory Activity of Pigment Echinochrome A
Ameliorative Effect of Novel Vitamin Formula with Herbal Extracts on Scopolamine-Induced Alzheimer's Disease
An Increase in Plasma Homovanillic Acid with Cocoa Extract Consumption Is Associated with the Alleviation of Depressive Symptoms in Overweight or Obese Adults
Attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal epithelium corresponds to disease severity