Amplite® Colorimetric Aldehyde Quantitation Kit Blue Color

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Amplite® Colorimetric Aldehyde Quantitation Kit

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. Kit 10053 uses a proprietary chromogenic dye that generates a strongly color product upon reacting with an aldehyde. This colorimetric kit provides a sensitive mix-and-read method to detect aldehydes (0.4 uM). 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.

Platform

Absorbance microplate reader

Absorbance	620 nm
Recommended plate	Clear bottom

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare Aldehyde standards and/or test samples (50 µL)
Add 2X AldeView™ Blue working solution (50 µL)
Incubate at RT for 20 minutes
Add AldeView™ Blue Enhancer (50 µL)
Incubate at RT for 20 minutes
Monitor absorbance increase at 620 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. Aldehyde standard solution (10 mM):
Add 1 mL of Assay Buffer (Component B) into the vial of Aldehyde Standard (Component D) to make a 10 mM Aldehyde standard solution.

PREPARATION OF STANDARD SOLUTION

Aldehyde standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/10053

Take 10 mM Aldehyde standard solution and perform 1:100 in Assay Buffer (Component B) to make 100 µM Aldehyde standard solution (AS7). Take 100 µM Aldehyde standard solution (AS7) and perform 1:2 serial dilutions to get serially diluted Aldehyde standards (AS6 - AS1) with Assay Buffer (Component B).

PREPARATION OF WORKING SOLUTION

Add 5 mL of Assay Buffer (Component B) into one bottle of AldeView™ Blue (Component A) to make 2X AldeView™ Blue working solution. Note: 5 mL of 2X AldeView™ Blue working solution is enough for one plate. 2X AldeView™ Blue working solution is not stable, and best used within 2 hours.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of Aldehyde Standards and test samples in a white 96-well microplate with clear bottom. AS= Aldehyde Standards (AS1 - AS7, 1.56 to 100 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 Dilutions (1.56 to 100 µM)
BL	50 µL	Assay Buffer
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 12.5 µL of reagent per well instead of 50 µL.
Add 50 µL of AldeView™ 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 12.5 µL of AldeView™ Blue working solution into each well instead, for a total volume of 25 µL/well.
Incubate the reaction at room temperature for 20 - 30 minutes, protected from light.
Add 50 µL of AldeView™ Blue Enhancer (Component C) into each well. For a 384-well plate, add 25 µL of AldeView™ Blue Enhancer into each well.
Incubate the reaction at room temperature for 20 minutes, protected from light.
Monitor the absorbance increase with an absorbance plate reader at around 620 to 660 nm (Max at 620 nm).

Images

Figure 1. Aldehyde dose response was measured in a white wall/clear bottom 96-well plate with Amplite Colorimetric Aldehyde Quantitation Kit using a SpectraMax microplate reader (Molecular Devices).

Figure 2. AldeView Blue (Component A) readily reacts with aldehydes to give AldeView Blue hydrazone which is dimerized in the presence of AldeView Blue enhancer (Component C). The dimer of AldeView Blue has strong absorption from 600 to 650 nm depending on the structures of aldehydes. The absorption of AldeView Blue dimer formed is proportional to the concentration of the aldehyde tested.

Citations

View all 5 citations: Citation Explorer

Aldehyde-mediated protein degradation is responsible for the inhibition of nucleotide excision repair by cigarette sidestream smoke
Authors: Yang, Guang and Komaki, Yukako and Ibuki, Yuko
Journal: Mutation Research/Genetic Toxicology and Environmental Mutagenesis (2018)

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

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