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|
|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-Mouse IgG-HRP Conjugate||1 vial (25 µL)|
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.
Open in Advanced Spectrum Viewer
|Name||Excitation (nm)||Emission (nm)|
|Amplite® Fluorimetric Goat Anti-Rabbit IgG-HRP Conjugate ELISA Assay Kit *Red Fluorescence*||571||584|
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