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

Protocol summary

1. Prepare D-lactate Dehydrogenase working solution (50 µL)
2. Add D-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 (Cutoff = 570nm)

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

2. Add 50 µL of 100X NAD stock solution into 5 mL Enzyme Probe mixture and mix well to make D-LDH working solution. Note: This D-LDH working solution is enough for one 96-well plate. It is not stable - make enough for one experiment and use promptly.

Table 1. Layout of D-LDH standards and test samples in a solid black 96-well microplate. SD=D-LDH Standards (SD1 - SD7, 0.3 to 300 mU/mL), BL=Blank Control, TS=Test Samples. 

Table 2. Reagent composition for each well.

1. Prepare D-LDH 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 D-LDH working solution to each well of D-LDH standard, blank control, and test samples to make the total D-LDH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of D-LDH working solution into each well instead, 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, Cutoff 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. The absorption detection has lower sensitivity compared to fluorescence reading.