Amplite® Fluorimetric Goat Anti-Mouse IgG-HRP Conjugate ELISA Assay Kit Red Fluorescence

Detection of mouse total IgG using the Amplite® Fluorimetric Goat Anti-Mouse IgG-HRP ELISA Kit. Mouse IgG was diluted into 3 µg/mL and made 1 to 3 serial dilutions in 0.2 M sodium bicarbonate buffer, pH 9.4. 100 µL/well serial dilutions were coated into a solid 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 by using the reagents. 1 to 5000 dilutions of goat anti-mouse IgG-HRP conjugate were used. The reactions were incubated for 10 to 60 minutes and then measured for fluorescence at Ex/Em = 540/590 nm using Gemini fluorescence microplate reader (Molecular Devices).

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
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Spectral properties

Excitation (nm)	571
Emission (nm)	584

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12171501

Overview SDS Protocol

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 monoclonal antibody/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 assays that detect goat anti-mouse IgG as the secondary detection agent.

Platform

Fluorescence microplate reader

Excitation	540 nm
Emission	590 nm
Cutoff	570 nm
Recommended plate	Solid black

Components

Example protocol

AT A GLANCE

Protocol summary

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

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). 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

Mouse IgG standard

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.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)	571
Emission (nm)	584

Product Family

Name	Excitation (nm)	Emission (nm)
Amplite® Fluorimetric Goat Anti-Rabbit IgG-HRP Conjugate ELISA Assay Kit Red Fluorescence	571	584

Images

Figure 1. Detection of mouse total IgG using the Amplite® Fluorimetric Goat Anti-Mouse IgG-HRP ELISA Kit. Mouse IgG was diluted into 3 µg/mL and made 1 to 3 serial dilutions in 0.2 M sodium bicarbonate buffer, pH 9.4. 100 µL/well serial dilutions were coated into a solid 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 by using the reagents. 1 to 5000 dilutions of goat anti-mouse IgG-HRP conjugate were used. The reactions were incubated for 10 to 60 minutes and then measured for fluorescence at Ex/Em = 540/590 nm using Gemini fluorescence microplate reader (Molecular Devices).

Citations

View all 11 citations: Citation Explorer

The combination of circRNA-UMAD1 and Galectin-3 in peripheral circulation is a co-biomarker for predicting lymph node metastasis of thyroid carcinoma
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

Assessment of Tofacitinib and Ruxolitinib and their Anti Inflammatory Effects on Myeloperoxidase
Authors: Milton, Amber
Journal: (2017)

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

References

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