Amplite® Fluorimetric Oxaloacetate Assay Kit *Red Fluorescence*

Protocol summary

1. Prepare Oxaloacetate standards or test samples (50 µL)
2. Add Oxaloacetate working solution (50 µL)
3. Incubate at room temperature for 30 - 60 minutes
4. Monitor fluorescence increase at Ex/Em = 540/590 nm (Cutoff = 570 nm)

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

1. Oxaloacetate standard solution (100 mM):
Add 100 µL of ddH2O into Oxaloacetate Standard (Component E) to make 100 mM Oxaloacetate standard solution.

2. Amplite™ Red substrate stock solution (200X):
Add 50 µL of DMSO (Component F) into Amplite™ Red substrate (Component A) to make 200X Amplite™ Red Substrate stock solution.

3. Oxaloacetate Decarboxcylase (OAC) stock solution (100X):
Add 50 µL of ddH2O into Oxaloacetate Decarboxcylase (Component D) to make 100X Oxaloacetate Decarboxcylase stock solution.

1. Add 5mL Assay Buffer (Component C) into one Enzyme Mix1 bottle (Component B1) and mix well.

2. Add 100 μL of ddH2O into one Enzyme Mix2 vial (Component B2) and mix well.

3. Transfer 25 μL of 200X Amplite™ Red stock solution, 50 μL of 100X OAC stock solution, and entire vial (100 μL) of Enzyme Mix2 into the Enzyme Mix1 bottle and mix well to make Oxaloacetate working solution. Note: This Oxaloacetate working solution is not stable, use it promptly, and avoid direct exposure to light.

Table 1. Layout of Oxaloacetate standards and test samples in a solid black 96-well microplate. SD=Oxaloacelate standard (SD1 - SD7, 0.1 to 100 µM), BL=Blank Control, TS=Test Samples. 

Table 2. Reagent composition for each well.

1. Prepare Oxaloacetate 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.
   
   
2. Add 50 µL of Oxaloacetate working solution to each well of Oxaloacetate standard, blank control, and test samples to make the total Oxaloacetate assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Oxaloacetate working solution into each well instead, for a total volume of 50 µL/well. Note: Run the Oxaloacetate assay at pH 6.5 to 7.0.
   
   
3. Incubate the reaction at room temperature for 30 - 60 minutes, protected from light.
   
   
4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm).