Amplite® Fluorimetric Malondialdehyde (MDA) Quantitation Kit
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12171501 |
Overview | SDSProtocol |
Platform
Fluorescence microplate reader
Excitation | 365 nm |
Emission | 435 nm |
Cutoff | 420nm |
Recommended plate | Solid black |
Components
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 notes
Thaw all the kit components to room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. 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.
2. MonoaldeliteTM Blue stock solution (250X):
Add 20 µL of DMSO (Component E) into one vial of MonoaldeliteTM Blue (Component A) to make MonoaldeliteTM Blue stock solution. Note: This MonoaldeliteTM Blue stock solution is enough for one 96-well plate. It is not stable, use it promptly.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/10071
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
MonoaldeliteTM Blue working solution:
Add 20 µL of 250X MonoaldeliteTM 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).
Images
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