Amplite® Fluorimetric Malondialdehyde (MDA) Quantitation Kit

Malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) are natural byproducts of lipid peroxidation. As the most popular and reliable biomarker for lipid peroxidation, MDA has been widely used for many years to determine oxidative stress in clinical situations. Therefore, quantification of MDA is essential to assess oxidative stress in pathophysiological processes. The Amplite® Fluorimetric Malondialdehyde (MDA) Quantitation Kit offers a quick and convenient method to measure MDA without heating steps that are required for the commercial MDA assay kits from other vendors. Monoaldelite™ Blue itself is nearly non-fluorescent, but generates strong blue fluorescence upon reacting with MDA.

Example protocol

AT A GLANCE

Protocol Summary

Prepare and add MDA standards or test samples (50 µL)
Prepare and add MDA working solution (50 µL)
Incubate at room temperature for 10 to 30 minutes
Add Reaction Solution (25 µL)
Incubate at room temperature for 30 to 60 minutes
Monitor fluorescence intensity at Ex/Em = 365/435 nm

Important Note

Thaw all the kit components to room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

MDA Standard Solution (100 mM)

Add 100 µL of ddH₂O into the vial of MDA Standard (Component C) to make 100 mM MDA standard solution.
Monoaldelite^TM Blue stock solution (250X):Add 20 µL of DMSO (Component E) into one vial of Monoaldelite^TM Blue (Component A) to make Monoaldelite^TM Blue stock solution.

Note: This Monoaldelite^TM Blue stock solution is enough for one 96-well plate. It is not stable, use it promptly.

PREPARATION OF STANDARD SOLUTIONS

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/10071

MDA standard

Add 10 µL of 100 mM MDA standard solution into 990 µL of Assay Buffer (Component B) to generate 1000 µM MDA standard solution (MDA7). Take 1000 µM MDA standard solution (MDA7) and perform 1:3 serial dilutions to get serially diluted MDA standards (MDA6 - MDA1) with Assay Buffer (Component B).

PREPARATION OF WORKING SOLUTION

Monoaldelite™ Blue working solution

Add 20 µL of 250X Monoaldelite^TM Blue stock solution into 5 mL of Assay Buffer (Component B) and mix them well.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of MDA standards and test samples in a solid black 96-well microplate. MDA= MDA Standards (MDA1 - MDA7, 1.37 to 1000 µM), BL=Blank Control, TS=Test Samples.

BL	BL	TS	TS
MDA1	MDA1	...	...
MDA2	MDA2	...	...
MDA3	MDA3
MDA4	MDA4
MDA5	MDA5
MDA6	MDA6
MDA7	MDA7

Table 2. Reagent composition for each well.

Well	Volume	Reagent
MDA1 - MDA7	50 µL	Serial Dilutions (1.37 to 1000 µM)
BL	50 µL	Assay Buffer (Component B)
TS	50 µL	test sample

Add MDA standards (MDA), 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 MDA working solution to each well of MDA 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 MDA working solution into each well instead, for a total volume of 50 µL/well.
Incubate the reaction at room temperature for 10 - 30 minutes.
Add 25 µL of Reaction Solution (Component D) to each well to make the total assay volume of 125 µL/well. For a 384-well plate, add 12.5 µL of Reaction Solution (Component D) into each well instead, for a total volume of 62.5 µL/well.
Incubate the reaction at room temperature for 30 - 60 minutes.
Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 365/435 nm (Cutoff = 420 nm).

Page updated on November 20, 2024

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