Amplite® Fluorimetric Malondialdehyde (MDA) Quantitation Kit Enhanced Selectivity

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Amplite® Fluorimetric Malondialdehyde (MDA) Quantitation Kit

Overview SDS Protocol

Malondialdehyde (MDA) is one of the natural byproducts during lipid peroxidation. It is widely used as a reliable biomarker to determine oxidative stress, and the quantification of MDA is an essential way to assess oxidative stress in pathophysiological processes. Amplite® Fluorimetric Malondialdehyde (MDA) Quantitation Kit offers a new method to measure MDA without the heating steps required for the TBARS-based MDA assay. The MDA Green™ can react with MDA to generate a considerable enhancement of green fluorescence signal in a convenient 96-well or 384-well microtiter-plate format. Unlike other commercial MDA assay kits, this assay is robust and specific to MDA with little interference from other aldehydes.

Platform

Fluorescence microplate reader

Excitation	480 nm
Emission	555 nm
Cutoff	530 nm
Recommended plate	Solid black

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare and add MDA standards or test samples (50 uL)
Prepare and add MDA Green™ working solution (50 uL)
Incubate at room temperature for 6 minutes
Add MDA Stopping Solution (50 uL)
Monitor fluorescence intensity at Ex/Em=480/555 nm

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

PREPARATION OF STOCK SOLUTION

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.

1. MDA standard solution (100 mM):
Add 100 uL of ddH₂O into the vial of MDA Standard (Component C) to make 100 mM MDA standard solution.

2. MDA Green™ stock solution (250 X):
Add 20 uL of DMSO (Component E) into one vial of MDA Green™ (Component A) to make vial of MDA Green™ stock solution. Note: All stock solutions should be stored at -20^oC after preparation. Avoid repeated freeze-thaw cycles.

PREPARATION OF STANDARD SOLUTION

MDA standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/10072

Add 10 uL of 100 mM MDA standard solution into 990 uL of MDA Assay Buffer (Component B) to generate 1000 uM MDA standard solution (MDA1). Take 1000 uM MDA standard solution (MDA1) and perform 1:3 serial dilutions to get serially diluted MDA standards (MDA2 - MDA7) with MDA Assay Buffer (Component B).

PREPARATION OF WORKING SOLUTION

MDA Green™ working solution:
Add 20 uL of 250 X MDA Green™ stock solution into 5 mL of MDA Assay Buffer (Component B), and mix well to make MDA Green™ working solution. Note: This MDA Green™ working solution should be prepared before the experiment, and kept from light.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of MDA standards and test samples in a solid black 96-well microplate. MDA= MDA Standards (MDA1 - MDA7, 1000 to 1.37 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 (1000 to 1.37 µM)
BL	50 µL	MDA Assay Buffer (Component B)
TS	50 µL	test sample

Prepare 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 uL of reagent per well instead of 50 uL.
Add 50 uL of MDA Green™ working solution to each well of MDA standard, blank control, and test samples. For a 384-well plate, add 25 uL of MDA Green™ working solution into each well instead.
Incubate the reaction at room temperature for 5-6 minutes, protected from light.
Add 50 uL of MDA Stopping Solution (Component D) to each well of the reaction. For a 384-well plate, add 25 uL of MDA Stopping Solution (Component D) into each well instead.
Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em=480/555 nm (Cutoff= 530 nm).

Images

Figure 1. MDA dose response was measured with Amplite® Fluorimetric Malondialdehyde (MDA) Quantitation Kit on a 96-well solid black microplate using a Gemini microplate reader (Molecular Devices) at Ex/Em=480/555 nm, cutoff=530 nm.

Figure 2. MDA selectivity against glyceraldehyde and formaldehyde were measured with Amplite® Fluorimetric Malondialdehyde (MDA) Quantitation Kit. MDA (2mM) showed high signal, while glyceraldehyde (2mM) or formaldehyde (2mM) has very little response to MDA Green™.

Citations

View all 91 citations: Citation Explorer

The Evaluation of Glutathione Reductase and Malondialdehyde Levels in Patients With Lumbar Disc Degeneration Disease
Authors: Bakirezer, S. D., Yaltirik, C. K., Kaya, A. H., Yilmaz, S. G., Ozdogan, S., Billur, D., Isbir, T.
Journal: In Vivo (2019): 811-814

Status of malondialdehyde, catalase and superoxide dismutase levels/activities in schoolchildren with iron deficiency and iron-deficiency anemia of Kashere and its environs in Gombe State, Nigeria
Authors: Sharif Usman, S., Dahiru, M., Abdullahi, B., Abdullahi, S. B., Maigari, U. M., Ibrahim Uba, A.
Journal: Heliyon (2019): e02214

Malondialdehyde Assay in the Evaluation of Aspirin Antiplatelet Effects
Authors: Polzin, A., Dannenberg, L., Schneider, T., Knoop, B., Naguib, D., Helten, C., Pohl, M., Kelm, M., Zeus, T., Hohlfeld, T.
Journal: Pharmacology (2019): 23-29

Glutathione peroxidase and malondialdehyde in children with chronic hepatitis C
Authors: Khedr, M. A., El-Araby, H. A., Konsowa, H. A., Sokar, S. S., Mahmoud, M. F., Adawy, N. M., Zakaria, H. M.
Journal: Clin Exp Hepatol (2019): 81-87

Malondialdehyde-acetaldehyde antibody concentrations in rheumatoid arthritis and other rheumatic conditions
Authors: Mikuls, T. R., Duryee, M. J., Engl and , B. R., Anderson, D. R., Hearth-Holmes, M., Su, K., Michaud, K., Payne, J. B., Sayles, H., Hunter, C., McGowan, J. D., Klassen, L. W., Thiele, G. M.
Journal: Int Immunopharmacol (2018): 113-118

Evaluation of correlation between expression of P53 and Malondialdehyde levels in prostate cancer patients
Authors: Arif, M., Rashid, A., Majeed, A., Qaiser, F., Razak, S.
Journal: J Pak Med Assoc (2018): 1373-1377

Estimation of malondialdehyde levels in serum and saliva of children affected with sickle cell anemia
Authors: Baliga, S., Chaudhary, M., Bhat, S., Bhansali, P., Agrawal, A., Gundawar, S.
Journal: J Indian Soc Pedod Prev Dent (2018): 43-47

Association between dietary and metabolic factors and IgM antibodies to phosphorylcholine and malondialdehyde in patients with systemic lupus erythematosus and population-based matched controls
Authors: Lourdudoss, C., Ajeganova, S., Frostegard, J.
Journal: Clin Exp Rheumatol (2018): 428-433

Elevated serum levels of malondialdehyde and cortisol are associated with major depressive disorder: A case-control study
Authors: Islam, M. R., Islam, M. R., Ahmed, I., Moktadir, A. A., Nahar, Z., Islam, M. S., Shahid, S. F. B., Islam, S. N., Islam, M. S., Hasnat, A.
Journal: SAGE Open Med (2018): 2050312118773953

Autoimmune reactivity to malondialdehyde adducts in systemic lupus erythematosus is associated with disease activity and nephritis
Authors: Hardt, U., Larsson, A., Gunnarsson, I., Clancy, R. M., Petri, M., Buyon, J. P., Silverman, G. J., Svenungsson, E., Gronwall, C.
Journal: Arthritis Res Ther (2018): 36