Amplite® Fluorimetric D-Lactate Dehydrogenase (LDH) Assay Kit
|Shipping||Standard overnight for United States, inquire for international|
|H-phrase||H303, H313, H333|
|Intended use||Research Use Only (RUO)|
|R-phrase||R20, R21, R22|
Fluorescence microplate reader
|Recommended plate||Solid black|
AT A GLANCE
- Prepare D-lactate Dehydrogenase working solution (50 µL)
- Add D-lactate Dehydrogenase standards or test samples (50 µL)
- Incubate at room temperature for 30 minutes - 2 hours
- Monitor fluorescence increase at Ex/Em = 540/590 nm (Cutoff = 570nm)
Thaw one of each kit component at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
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.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13808
Add 10 µL of 100 U/mL D-LDH standard solution into 990 µL 1x PBS buffer to generate 1000 mU/mL D-LDH standard solution. Take 1000 mU/mL D-LDH standard solution and perform 1:3 serial dilutions in PBS to get serial dilutions of D-LDH standard (SD7 - SDH1). Note: Diluted D-LDH standard solution is unstable, and should be used within 4 hours.
PREPARATION OF WORKING 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.
SAMPLE EXPERIMENTAL PROTOCOL
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.
|SD1 - SD7||50 µL||Serial Dilutions (0.3 to 300 mU/mL)|
|BL||50 µL||Dilution Buffer|
|TS||50 µL||test sample|
- 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.
- 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.
- Incubate the reaction at room temperature for 30 minutes to 2 hours, protected from light.
- 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 A575nm/A605nm. The absorption detection has lower sensitivity compared to fluorescence reading.
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