Amplite® Fluorimetric Glutamic Acid Assay Kit *Red Fluorescence*

Protocol summary

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

Important notes
Thaw all the kit components at room temperature before starting the experiment.

1. NADP stock solution (200X):
Add 100 µL of Dilution Buffer (Component E) into the vial of NADP (Component C) to make 200X NADP stock solution. 

2. Glutamic Acid standard solution (100 mM):
Add 200 µL of Dilution Buffer (Component E) into the vial of Glutamic Acid (Component D) to make 100mM Glutamic Acid standard solution. 

1. Add 10 mL of Assay Buffer (Component B) into the bottle of Enzyme Mix (Component A).

2. Add 50 µL 200X NADP stock solution into the Enzyme Mix bottle, and mix well to make Glutamic Acid working solution.  Note: This Glutamic Acid working solution is enough for two 96-well plates. It is unstable at room temperature, and should be used promptly within 2 hours and avoid exposure to light.  Note: Alternatively, one can make a 50X of Enzyme Mix stock solution by adding 200 μL of H2O into the bottle of Enzyme Mix (Component A), and then prepare the Glutamic Acid working solution by mixing the stock solution with Assay Buffer (Component B) and 200X NADP stock solution proportionally.

Table 1. Layout of Glutamic Acid standards and test samples in a solid black 96-well microplate.  SD = Glutamic Acid Standard, BL = Blank Control, TS = Test Sample. 

Table 2. Reagent composition for each well

1. Prepare Glutamic Acid 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 Glutamic Acid working solution into each well of Glutamic Acid standard, blank control, and test samples to make the total Glutamic Acid assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Glutamic Acid working solution into each well intead, for the total volume of 50 µL/well.
   
   
3. Incubate the reaction at room temperature for 30 minutes to 2 hours, protected from light.
   
   
4. Monitor the fluorescence increase with a fluorescence plate reader at  Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm), Cutoff = 570 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 absorbance ratio of ~570 nm to ~605 nm (A575nm/A605nm). The absorption detection has lower sensitivity compared to the fluorescence reading.