Amplite® Fluorimetric L-Lactate Dehydrogenase (LDH) Assay Kit

Protocol summary

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

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

1. NAD stock solution (100X):
Add 100 µL of H₂O into the vial of NAD (Component C) to make 100X NAD stock solution.

2. L-LDH standard solution (100 U/mL):
Add 100 µL of H₂O or 1X PBS buffer into the vial of L-LDH standard (Component D) to make 100 U/mL L-LDH standard solution.

1. Add 10 mL of Assay Buffer (Component B) into the bottle of Enzyme Probe (Component A) to have enzyme probe mixture. Note: This enzyme probe mixture is enough for two 96-well plates.

2. Add 50 µL NAD stock solution (100X) into 5 mL of enzyme probe mixture, and mix well. Note: This L-LDH working solution is not stable, and should be used promptly.

Table 1. Layout of L-LDH standards and test samples in a solid black 96-well microplate. L-LDH = L-LDH standard (L-LDH1 - L-LDH7, 0.3 to 300 mU/mL); BL = blank control; TS = test sample.

 Table 2. Reagent composition for each well.

1. Prepare L-LDH standards (L-LDH), blank controls (BL), and test samples (TS) 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.
   
   
2. Add 50 µL of L-LDH working solution to each well of L-LDH standard, blank control, and test samples to make the total assay volume of 100 µL/well. For a 384-well plate, add 25 µL of working solution into each well isntead, for a 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, cut off at 570 nm). Note: The contents of the plate can also be transferred to a white/clear bottom plate and read by absorbance microplate reader at the ratio of A_575nm/A_605nm. However, the absorption detection will have a lower sensitivity compared to that of the fluorescence reading.