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

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.

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.

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

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.