Amplite™ Red

Protocol summary for Peroxidase (HRP) with Amplite^TM Red (for one 96 well black plate)

1. Prepare and add 1X Amplite^TM Red working solution with 200 mM H₂O₂ in phosphate buffer (50 µL)
2. Add Peroxidase standards or test samples (50 µL)
3. Incubate at room temperature for 10-30 minutes
4. Monitor fluorescence intensity at Ex/Em = 540/590 nm

Important notes
The following is the recommended protocol for peroxidase assay in solution. The protocol only provides a guideline, should be modified according to the specific needs.

Thaw one of each kit component at room temperature before starting the experiment.

Protocol summary for H₂O₂ with Amplite^TM Red (for one 96 well black plate)

1. Prepare 1X Amplite^TM Red H₂O₂ working solution with 0.4 U/mL peroxidase in phosphate buffer (50 µL)
2. Add Peroxidase standards or test samples (50 µL)
3. Incubate at room temperature for 10-30 minutes
4. Monitor fluorescence intensity at Ex/Em = 540/590 nm

Important notes
The following is the recommended protocol for H₂O₂ assay in solution. The protocol only provides a guideline, should be modified according to the specific needs.

Thaw one of each kit component at room temperature before starting the experiment.

Amplite^TM Red stock solution (250X):
Add 200 mL of anhydrous DMSO into the vial, mixed well. The stock solution should be used promptly. Any unused solution need to be aliquoted and refrozen at < -20 ^o. Note: Avoid repeated freeze-thaw cycles, and protect from light.

1. Amplite^TM Red Peroxidase working solution (1X):
Add 20 μL of Amplite^TM Red stock solution (250X) in 5 mL of 50 mM phosphate buffer or buffer of your choice, pH 7 with 200 mM H₂O₂. Note: Amplite^TM Red is unstable in the presence of thiols such as DTT and b-mercaptoethanol. Thiols higher than 10 μM (final concentration) could significantly decrease the assay dynamic range. NADH and glutathione (reduced from: GSH) may interfere with the assay.

2. Amplite^TM Red H₂O₂ working solution (1X):
Add 20 μL of Amplite^TM Red stock solution (250X) in 5 mL of 50 mM phosphate buffer or buffer of your choice, pH 7 with 0.4 units/mL peroxidase. Note: Amplite^TM Red is unstable in the presence of thiols such as DTT and b-mercaptoethanol. Thiols higher than 10 μM (final concentration) could significantly decrease the assay dynamic range. NADH and glutathione (reduced from: GSH) may interfere with the assay.

Peroxidase assay in supernatants

1. Add 50 µL of 1X Amplite^TM Red peroxidase working solution into each well of the peroxidase standard, blank control, and test samples to make the total peroxidase assay volume of 100 µL/well. Note: For a 384-well plate, add 25 µL of sample and 25 µL of 1X Amplite^TM Red peroxidase working solution into each well.
   
   
2. Incubate the reaction at room temperature for 10 to 30 minutes, protected from light.
   
   
3. Monitor the fluorescence increase at Ex/Em = 540/590 nm with a fluorescence plate reader.
   
   
4. The fluorescence in blank wells (with the assay buffer only) is used as a control, and is subtracted from the values for those wells with the peroxidase reactions.

H₂O₂ assay in supernatants

1. Add 50 µL of 1X Amplite^TM Red H₂O₂ working solution into each well of the H₂O₂ standard, blank control, and test samples to make the total H₂O₂ assay volume of 100 µL/well. Note: For a 384-well plate, add 25 µL of sample and 25 µL of 1X Amplite^TM Red H₂O₂ working solution into each well.
   
   
2. Incubate the reaction at room temperature for 10 to 30 minutes, protected from light.
   
   
3. Monitor the fluorescence increase at Ex/Em = 540/590 nm with a fluorescence plate reader.
   
   
4. The fluorescence in blank wells (with the assay buffer only) is used as a control, and is subtracted from the values for those wells with the H₂O₂ reactions.

The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate HRP samples. We recommend using the Online Linear Regression Calculator which can be found at:

https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator

Figure 1. HRP dose response was measured with Amplite™ Fluorimetric Peroxidase Assay Kit in a black plate using a Gemini fluorescence microplate reader (Molecular Devices).