荧光法过氧化物酶(辣根过氧化物酶)检测试剂盒

Protocol summary

1. Prepare HRP working solution (50 µL)
2. Add HRP standards and/or test samples (50 µL)
3. Incubate at room temperature for 10 - 30 minutes
4. Monitor fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 575 nm)

Important notes
Thaw all the kit components at room temperature before starting the experiment. The component A is unstable in the presence of thiols such as DTT and β-mercaptoethanol. The presence of thiols at concentration higher than 10 µM would significantly decrease the assay dynamic range. NADH and glutathione (reduced form: GSH) may interfere with the assay.

1. Amplite™ Red Peroxidase Substrate stock solution (100X):
Add 250 µL of DMSO (Component E) into the vial of Amplite™ Red Peroxidase Substrate (Component A) to make 100X Amplite™ Red Peroxidase Substrate stock solution. The stock solution should be used promptly. Keep from light.

2. HRP standard solution (20 U/mL):
Add 1 mL of Assay Buffer (Component C) into the vial of Horseradish Peroxidase (Component D) to make 20 U/mL of HRP standard solution.

3. 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) to make 20 mM H₂O₂ stock solution . Note: The diluted H₂O₂ solution is not stable. The unused portion should be discarded.

Add 1 µL of 20 U/mL HRP standard solution in 1999 µL of Assay Buffer (Component C) to get 10 mU/mL HRP standard solution (SD7). Take 10 mU/mL HRP standard solution (SD7) and perform 1:3 serial dilutions to get serially diluted HRP standards (SD6 - SD1) with Assay Buffer (Component C).

Add 50 μL of 100X Amplite™ Red Peroxidase Substrate stock solution and 50 μL of 20 mM H₂O₂ stock solution into 4.9 mL of Assay Buffer (Component C) to make HRP working solution. Keep from light.

Table 1. Layout of HRP standards and test samples in a solid black 96-well microplate. SD= HRP Standards (SD1 - SD7, 0.01 to 10 mU/mL); BL=Blank Control; TS=Test Samples.

Table 2. Reagent composition for each well.

1. Prepare HRP standards (SD), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL. Note: High levels of HRP (e.g., >100 mU/mL final concentration) may cause reduced fluorescence signal due to the over oxidation of Amplite™ Red (to non-fluorescent one).
   
2. Add 50 µL of HRP working solution to each well of HRP standard, blank control, and test samples to make the total HRP assay volume of 100 µL/well. For a 384-well plate, add 25 µL of HRP working solution into each well instead, for a total volume of 50 µL/well.
   
3. Incubate the reaction at room temperature for 10 to 30 minutes, protected from light.
   
4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 540 ± 10 nm, Emission = 590 ± 10 nm (optimal Ex/Em = 540/590 nm, Cutoff = 575 nm). Note: The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 576 ± 5 nm. The absorption detection has lower sensitivity compared to fluorescence reading.