Amplite® Red

1. Prepare and add 1X Amplite® Red working solution with 200 µM 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

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.

1. Prepare 1X Amplite® 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

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.

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

Add 200 µL of anhydrous DMSO into the vial and mix well. The stock solution should be used promptly. Any unused solution needs to be aliquoted and refrozen at <-20°C.

Note: Avoid repeated freeze-thaw cycles and protect from light.

Add 20 μL of Amplite® Red stock solution (250X) in 5 mL of 50 mM phosphate buffer or buffer of your choice, pH 7, with 200 μM H₂O₂.

Note: Amplite® 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.

Add 20 μL of Amplite® 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® 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.

1. Add 50 µL of 1X Amplite® 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® 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.

1. Add 50 µL of 1X Amplite® 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® 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.