Amplite® Fluorimetric Renin Assay Kit *Red Fluorescence*

Protocol summary

1. Add appropriate controls, or test samples (50 µL)
2. Add Renin Red™ substrate working solution (50 µL)
3. Incubate for 30 - 60 min at 37 °C incubator (for end point reading)
4. Monitor fluorescence intensity at Ex/Em = 540/590 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment. Prepare Renin containing biological samples as desired.

Add 50 μL of Renin Red™ Substrate (Component A) into 5 mL of Assay Buffer (Component C) to make a total volume of 5.05 mL. Note: The Renin Red™ Substrate should be used promptly.

Table 1. Layout of Renin standards and test samples in a solid black 96-well microplate. Ren = Renin Standards (Ren1-Ren7, 1 to 1000 ng/mL); BL = Blank Control; TS = Test Samples.

Table 2. Reagent composition for each well. Note: The Renin standards are for positive control only, and should not be relied on as a quantitation standard for enzyme activity.

1. Prepare Renin containing biological samples as desired.
   
   
2. Prepare the Renin standards and/or Renin-containing test samples according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
   
   
3. Pre-incubate the plate at a desired temperature for the enzyme reaction (e.g. 25 °C or 37 °C) for 10 - 15 min, if you are screening Renin inhibitors.
   
   
4. Add 50 µL (96-well) or 25 µL (384-well) of Renin Red™ substrate working solution to the sample and control wells of the assay plate.
   
   
5. Incubate the reaction at 37 °C incubator for 30 to 60 minutes.
   
   
6. Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em = 540/590 nm (cut off = 570 mn). Note: The selectivity of the renin substrate used in the kit has not been thoroughly tested. It may also respond to other proteases since peptide-based protease substrates generally have low selectivity. One might use a renin-specific inhibitor for its specific test, such as in the presence of a renin-specific inhibitor, hydrolysis of the substrate is only due to the non-specific protease activity. The difference between the total activity and the activity in the presence of renin specific Inhibitor gives the renin activity in the sample.
   
   For kinetic reading: Immediately start measuring fluorescence intensity and continuously record data every 5 minutes for 30 to 60 minutes.
   
   For end-point reading: Incubate the reaction at 37 °C for 60 minutes or longer, kept from light if possible. And then measure the fluorescence intensity.