Amplite® Fluorimetric Aldehyde Quantitation Kit

Ethanol-induced changes in the gene expression of alcohol-metabolizing enzymes in WT and ALR-deficient mice. Hepatic acetaldehyde concentration. *Hepatic acetaldehyde was measured using Amplite® Fluorimetric Aldehyde Quantitation kit (10052) (AAT Bioquest, Inc., Sunnyvale, CA). Source: Graph from <strong>Hepatic Deficiency of Augmenter of Liver Regeneration Exacerbates Alcohol-Induced Liver Injury and Promotes Fibrosis in Mice</strong> by Sudhir Kumar, et al., <em>PLOS ONE</em>, Jan. 2016.

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H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12171501

Overview SDS Protocol

The formation, reactivity and toxicity of aldehydes originating from the peroxidation of lipids of cellular membranes have received great attention in recent years. Rapid and accurate measurement of aldehydes is an important task for biological research, chemical research, food industry and environmental pollution surveillance. There are a few reagents or assay kits available for quantifying the number of aldehydes. Most of the existing aldehyde test methods are based on separations either by the tedious and expensive HPLC-MS or GC-MS. Both our Amplite® Colorimetric Aldehyde Quantitation Kit (10051) and Amplite® Fluorimetric Aldehyde Quantitation kit (10052) are used for quantifying aldehydes at higher pH. Kit 10052 uses a proprietary fluorogenic dye that generates a strongly fluorescent product upon reacting with an aldehyde. Kit 10052 is much more sensitive than Kit 10051. This fluorimetric kit provides a sensitive mix-and-read method to detect as little as 0.1 nanomole of aldehyde in a 100 µL assay volume (1 µM). The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. Its signal can be easily read by a fluorescence microplate reader.

Platform

Fluorescence microplate reader

Excitation	365
Emission	435
Cutoff	420
Recommended plate	Solid black

Components

Example protocol

AT A GLANCE

Protocol Summary

Prepare Aldehyde Standards and/or test samples (50 µL)
Add AldeLight™ Blue working solution (50 µL)
Incubate at RT for at least 30 minutes
Add 25 µL of Reaction Buffer
Monitor fluorescence increase at Ex/Em = 365/435 nm

Important 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.

1. AldeLight™ Blue stock solution (250X)

Add 40 µL of DMSO (Component E) into the vial of AldeLight™ Blue (Component A) to make 250X AldeLight™ Blue stock solution.

2. Aldehyde standard solution (10 mM)

Add 1 mL of ddH₂O into the vial of Aldehyde Standard (Component D) to make a 10 mM aldehyde standard solution.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/10052

Aldehyde standard

Take 10 mM aldehyde standard solution and perform 1:10 dilutions in ddH2O to get 1000 uM Aldehyde standard solution (AS7). Then perform 1:3 serial dilutions to get remaining aldehyde standard (AS6 - AS1).

PREPARATION OF WORKING SOLUTION

Add 20 μL of 250X AldeLight™ Blue stock solution into 5 mL of Assay Buffer (Component B), and mix well to make AldeLight™ Blue working solution.
Note 5 mL of AldeLight™ Blue working solution is enough for one plate. The reaction mixture is not stable, and best used within 2 hours.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of Aldehyde Standards and test samples in a solid black 96-well microplate. AS= Aldehyde Standards (AS1 - AS7, 1 to 1000 µM); BL= Blank Control; TS= Test Samples.

BL	BL	TS	TS
AS1	AS1	...	...
AS2	AS2	...	...
AS3	AS3
AS4	AS4
AS5	AS5
AS6	AS6
AS7	AS7

Table 2. Reagent composition for each well.

Well	Volume	Reagent
AS1 - AS7	50 µL	Serial Dilution (1 to 1000 µM)
BL	50 µL	ddH₂O
TS	50 µL	test sample

Prepare Aldehyde standards (AS), 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 AldeLight™ Blue working solution to each well of aldehyde standard, blank control, and test samples to make the total aldehyde assay volume of 100 µL/well. For a 384-well plate, add 25 µL of AldeLight™ Blue working solution into each well instead, for a total volume of 50 µL/well.
Incubate the reaction mixture at room temperature for 30 minutes or more, protected from light.
Add 25 µL of Reaction Buffer (Component C) into each well.
Monitor the fluorescence increase at Ex/Em = 365/435 nm using a fluorescence plate reader.

Images

Figure 1. Aldehyde dose response was measured in a solid black 96-well plate with Amplite® Fluorimetric Aldehyde Quantitation Kit using a Gemini fluorescence microplate reader (Molecular Devices).

Figure 2. Ethanol-induced changes in the gene expression of alcohol-metabolizing enzymes in WT and ALR-deficient mice. Hepatic acetaldehyde concentration. *Hepatic acetaldehyde was measured using Amplite® Fluorimetric Aldehyde Quantitation kit (10052) (AAT Bioquest, Inc., Sunnyvale, CA). Source: Graph from Hepatic Deficiency of Augmenter of Liver Regeneration Exacerbates Alcohol-Induced Liver Injury and Promotes Fibrosis in Mice by Sudhir Kumar, et al., PLOS ONE, Jan. 2016.

Citations

View all 7 citations: Citation Explorer

Liver regeneration and ethanol detoxification: A new link in YAP regulation of ALDH1A1 during alcohol-related hepatocyte damage
Authors: Zhou, Junmei and Sun, Chunbao and Yang, Lu and Wang, Jinhui and Jn-Simon, Natacha and Zhou, Chen and Bryant, Andrew and Cao, Qi and Li, Chenglong and Petersen, Bryon and others,
Journal: The FASEB Journal (2022): e22224

Inhibition of ALDH2 by O-GlcNAcylation contributes to the hyperglycemic exacerbation of myocardial ischemia/reperfusion injury
Authors: Liu, Baoshan and Wang, Jiali and Li, Minghua and Yuan, Qiuhuan and Xue, Mengyang and Xu, Feng and Chen, Yuguo
Journal: Oncotarget (2017): 19413

Biological Activity of Peptide-conjugated Polyion Complex Matrices Consisting of Alginate and Chitosan
Authors: Fujimori, Chikara and Kumai, Jun and Nakamura, Kyotaro and Gu, Yingzi and Katagiri, Fumihiko and Hozumi, Kentaro and Kikkawa, Yamato and Nomizu, Motoyoshi
Journal: Peptide Science (2016)

Integrated self-assembling drug delivery system possessing dual responsive and active targeting for orthotopic ovarian cancer theranostics
Authors: Lin, Chun-Jui and Kuan, Chen-Hsiang and Wang, Li-Wen and Wu, Hsi-Chin and Chen, Yunching and Chang, Chien-Wen and Huang, Rih-Yang and Wang, Tzu-Wei
Journal: Biomaterials (2016): 12--26

Hepatic Deficiency of Augmenter of Liver Regeneration Exacerbates Alcohol-Induced Liver Injury and Promotes Fibrosis in Mice
Authors: Kumar, Sudhir and Wang, Jiang and Rani, Richa and G, undefined and hi, Ch and rashekhar R, undefined
Journal: PloS one (2016): e0147864

Einsatz funktioneller Magnetpartikel in der Biokatalyse: Enzymimmobilisierung, Charakterisierung und Optimierung
Authors: Morhardt, Christian
Journal: (2015)

Fiber-optic protease sensor based on the degradation of thin gelatin films
Authors: Schyrr, Bastien and Boder-Pasche, Stéphanie and Ischer, Réal and Smajda, Rita and Voirin, Guy
Journal: Sensing and Bio-Sensing Research (2015): 65--73