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Amplite™ Fluorimetric Goat Anti-Rabbit IgG-HRP Conjugate ELISA Assay Kit *Red Fluorescence*

Detection of rabbit total IgG using the Amplite™ Fluorimetric ELISA Kit. Rabbit IgG was diluted into 1 µg/mL and made 1 to 3 serial dilutions in 0.2 M sodium bicarbonate buffer at pH 9.4. 100 µL/well serial dilutions were coated into a black 96-well plate at 4°C overnight, and blocked with 3% milk in PBS and 0.02% Tween 20 at 4°C overnight. The wells were washed, and assayed using the reagents. 1 to 6000 dilutions of goat anti-rabbit IgG-HRP conjugate were used. The reactions were incubated for 15 to 60 minutes and then measured for fluorescence at Ex/Em = 540/590 nm with Gemini fluorescence microplate reader (Molecular Devices).
Detection of rabbit total IgG using the Amplite™ Fluorimetric ELISA Kit. Rabbit IgG was diluted into 1 µg/mL and made 1 to 3 serial dilutions in 0.2 M sodium bicarbonate buffer at pH 9.4. 100 µL/well serial dilutions were coated into a black 96-well plate at 4°C overnight, and blocked with 3% milk in PBS and 0.02% Tween 20 at 4°C overnight. The wells were washed, and assayed using the reagents. 1 to 6000 dilutions of goat anti-rabbit IgG-HRP conjugate were used. The reactions were incubated for 15 to 60 minutes and then measured for fluorescence at Ex/Em = 540/590 nm with Gemini fluorescence microplate reader (Molecular Devices).
Ordering information
Price ()
Catalog Number11541
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
Spectral properties
Excitation (nm)571
Emission (nm)584
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

OverviewpdfSDSpdfProtocol


Excitation (nm)
571
Emission (nm)
584
Horseradish Peroxidase (HRP) is a small molecule (MW ~40 KD) that is widely used in a variety of biological detections. HRP conjugates are extensively used as secondary detection reagents in ELISAs, immuno-histochemical techniques, Northern, Southern and Western blot analyses. Due to its small size, it rarely causes steric hindrance problem with antibody/antigen complex formation. It is usually conjugated to an antibody in a 4:1 ratio. Additionally, HRP is inexpensive compared to other labeling enzymes. The major disadvantage associated with peroxidase is their low tolerance to many preservatives such as sodium azide that inactivates peroxidase activity even at low concentration. Our Amplite™ Fluorimetric ELISA Assay Kit contains all the essential components including our fluorogenic Amplite™ Red HRP substrate for ELISA detection. The kit provides an optimized assay protocol that is compatible with HTS liquid handling, as little as 10 pg of a Polyclonal antibody in the well of a microplate can be detected. Its signal can be easily read by either fluorescence microplate reader or absorbance microplate reader. It can be used for the assays that detect goat anti-rabbit IgG as the secondary detection agent.

Platform


Absorbance microplate reader

Recommended plateClear bottom

Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black

Components


Component A: Amplite™ Red Peroxidase Substrate2 vials
Component B: H2O21 vial (3% stabilized solution, 500 µL)
Component C: Assay Buffer1 bottle (100 mL)
Component D: DMSO1 vial (1 mL)
Component E: Goat Anti-Rabbit IgG-HRP Conjugate1 vial (25 µL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare ELISA plate
  2. Add Goat Anti-Rabbit IgG-HRP Conjugate working solution (100 µL/well)
  3. Incubate at room temperature for 30 minutes
  4. Wash the wells (3X with PBS-Tween)
  5. Add Peroxidase working solution (100 µL/well) 
  6. Incubate at room temperature for 30 - 60 minutes 
  7. Monitor fluorescence intensity at Ex/Em = 540/490 nm (Cutoff = 570 nm)

Important notes
Thaw all the kit components at room temperature before starting the experiment.

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.

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
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

Prepare ELLISA plate:

  1. Prepare ELISA microplate (including appropriate controls) by performing all necessary ELISA preparation steps.

  2. 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.

  3. Wash the ELISA wells three times with PBS containing 0.02% to 0.05% Tween® 20 (PBS-Tween) and drain.

  4. Add 100 µL of Goat Anti-Rabbit IgG-HRP Conjugate working solution into each well.

  5. Incubate at room temperature for 30 minutes. Drain off the Goat Anti-Rabbit IgG-HRP Conjugate.

  6. Wash the wells three times with PBS-Tween and drain.

Run Peroxidase assay in ELISA plate:

  1. Add 100 µL of Peroxidase working solution into each drained microplate well containing the samples and controls.

  2. Incubate the reaction at room temperature for 30 minutes or longer, protected from light.

  3. 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.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)571
Emission (nm)584

Citations


View all 10 citations: Citation Explorer
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
Distinguishing inflammation from tumor and peritumoral edema by myeloperoxidase magnetic resonance imaging
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

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