Amplite® Colorimetric α-Ketoglutarate Quantitation Kit

Protocol summary

1. Prepare test samples along with serially diluted α-ketoglutarate standards (50 µL)
2. Add equal volume of α-Ketoglutarate working solution (50 µL)
3. Incubate at 37°C for 60 - 90 minutes
4. Monitor absorbance intensity at 570 nm

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

1. Amplite™ Red stock solution (200X):
Add 50 µL of DMSO (Component E) into the vial of Amplite™ Red (Component A) to make 200X stock solution.

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

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

3. Transfer entire vial (100 μL) of Enzyme Mix 2 and 25 µL of 200X Amplite™ Red stock solution into the vial of Enzyme Mix 1 and mix well to make α-Ketoglutarate working solution. Note: The 5 mL α-Ketoglutarate working solution is enough for one 96-well plate. It is not stable, use it promptly.

Table 1. Layout of α-ketoglutarate standards and test samples in a 96-well clear bottom microplate. AKG= α-Ketoglutarate Standard (AKG1 - AKG7, 1.563 to 100 µM), BL=Blank Control, TS=Test Sample. 

Table 2. Reagent composition for each well.

1. Prepare α-ketoglutarate standards (AKG), 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 α-Ketoglutarate working solution to each well of α-ketoglutarate standard, blank control, and test samples to make the total α-ketoglutarate assay volume of 100 µL/well. For a 384-well plate, add 25 µL of α-Ketoglutarate working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction mixture at 37°C for 60 - 90 minutes.
   
   
4. Monitor the absorbance increase with an absorbance plate reader with path check on at OD of 570 nm.