Amplite® Fluorimetric Goat Anti-Rabbit 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|
Absorbance microplate reader
|Recommended plate||Clear bottom|
Fluorescence microplate reader
|Recommended plate||Solid black|
|Component A: Amplite™ Red Peroxidase Substrate||2 vials|
|Component B: H2O2||1 vial (3% stabilized solution, 500 µL)|
|Component C: Assay Buffer||1 bottle (100 mL)|
|Component D: DMSO||1 vial (1 mL)|
|Component E: Goat Anti-Rabbit IgG-HRP Conjugate||1 vial (25 µL)|
AT A GLANCE
- Prepare ELISA plate
- Add Goat Anti-Rabbit IgG-HRP Conjugate working solution (100 µL/well)
- Incubate at room temperature for 30 minutes
- Wash the wells (3X with PBS-Tween)
- Add Peroxidase working solution (100 µL/well)
- Incubate at room temperature for 30 - 60 minutes
- Monitor fluorescence intensity at Ex/Em = 540/490 nm (Cutoff = 570 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) and mix well to make 200X Amplite™ Red Peroxidase Substrate stock solution. Note: 50 µL of 200X Amplite™ Red Peroxidase Substrate stock solution is enough for 1 plate. The stock solution should be used promptly. Protect from light.
2. H2O2 stock solution (20 mM):
Add 22.7 µL of 3% H2O2 (0.88 M, Component B) into 977µL of Assay Buffer (Component C) to make 20 mM H2O2 stock solution. Note: The diluted H2O2 stock solution is not stable. The unused portion should be discarded.
PREPARATION OF WORKING SOLUTION
Add 50 μL of 200X Amplite™ Red Peroxidase Substrate stock solution and 100 μL of 20 mM H2O2 stock solution into 9.85 mL of Assay Buffer (Component C) and mix well to make Peroxidase working solution. Keep from light.
For guidelines on cell sample preparation, please visit
SAMPLE EXPERIMENTAL PROTOCOL
Prepare ELLISA plate:
- Prepare ELISA microplate (including appropriate controls) by performing all necessary ELISA preparation steps.
- Add 2 µL of Goat Anti-Rabbit IgG-HRP Conjugate (Component E) into 10 mL of PBS with 1% BSA (PBS-BSA, not included) to make Goat Anti-Rabbit IgG-HRP Conjugate working solution. Note: 10 mL of Goat Anti-Rabbit IgG-HRP Conjugate working solution is enough for 1 plate. The concentration of this Goat Anti-Rabbit IgG-HRP Conjugate working solution is recommended as an initial concentration to try. The optimal concentration for each particular application may need to be determined empirically.
- Wash the ELISA wells three times with PBS containing 0.02% to 0.05% Tween® 20 (PBS-Tween) and drain.
- Add 100 µL of Goat Anti-Rabbit IgG-HRP Conjugate working solution into each well.
- Incubate at room temperature for 30 minutes. Drain off the Goat Anti-Rabbit IgG-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, Emission = 590-600 nm (optimal Ex/Em = 540/590 nm, Cutoff = 570 nm). Note: The plate can also be read by an absorbance microplate reader at the wavelength of 576 ± 5 nm. The absorption detection has lower sensitivity compared to fluorescence reading.
Open in Advanced Spectrum Viewer
|Name||Excitation (nm)||Emission (nm)|
|Amplite® Fluorimetric Goat Anti-Mouse IgG-HRP Conjugate ELISA Assay Kit *Red Fluorescence*||571||584|
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: 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
Journal: Clinical Cancer Research (2011): 4484--4493
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)
A Library of Well-Defined and Water-Soluble Poly(alkyl phosphonate)s with Adjustable Hydrolysis
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