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Amplite® Colorimetric Glycerol Assay Kit

Glycerol dose response was measured with Amplite® Colorimetric Glycerol Assay Kit on a black wall/clear bottom 96-well plate using a SpectraMax reader.
Glycerol dose response was measured with Amplite® Colorimetric Glycerol Assay Kit on a black wall/clear bottom 96-well plate using a SpectraMax reader.
Glycerol dose response was measured with Amplite® Colorimetric Glycerol Assay Kit on a black wall/clear bottom 96-well plate using a SpectraMax reader.
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H-phraseH303, H313, H333
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Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Glycerol is a precursor for synthesis of triglycerides and phospholipids in the liver and adipose tissue. When fasting, triglycerides stored in these lipid droplets can be hydrolyzed to generate free glycerol and fatty acids. The amount of free glycerol released to the bloodstream is proportional to the triglyceride/fatty acid cycling rate, which is important in the metabolic regulation and heat production. Amplite® Colorimetric Glycerol Assay Kit offers a sensitive assay for measuring glycerol levels in biological samples. This assay is based on an enzyme coupled reaction of glycerol, in which the product hydrogen peroxide can be detected using our Amplite® Red HRP substrate in the HRP-coupled reactions. The signal can be measured with an absorbance microplate reader using OD 575 nm. With this Colorimetric Glycerol Assay Kit, we were able to detect as low as 0.15 µg/mL (~1.6 µM) glycerol in a 100 µL reaction volume.

Platform


Absorbance microplate reader

Absorbance575 nm
Recommended plateClear bottom
Instrument specification(s)Path check

Components


Example protocol


AT A GLANCE

Protocol Summary
  1. Prepare glycerol standards or test samples (50 µL)
  2. Add glycerol working solution (50 µL)
  3. Incubate at room temperature for 30 min to 1 hour
  4. Monitor OD at 575 nm
Important Note

To achieve the best results, it’s strongly recommended to use the black plates. Thaw one vial of each kit component at 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

Amplite™ HRP substrate stock solution (200X)

Add 50 µL of DMSO (Component E) into the vial of Amplite™ HRP substrate (Component A) to make 200X stock solution. Note: The Amplite™ Red is unstable in the presence of thiols such as dithiothreitol (DTT) and 2-mercaptoethanol. The final concentration of DTT or 2-mercaptoethanol in the reaction should be no higher than 10 µM. The Amplite™ Red is also unstable at high pH (>8.5). Therefore, the reaction should be performed at pH 7 – 8. The provided assay buffer (pH 7.4) is recommended.

Glycerol standard solution

Add 1 mL of ddH2O or 1X PBS buffer into the vial of glycerol standard (Component D) to make 1 mg/mL glycerol standard solution.

PREPARATION OF STANDARD SOLUTIONS

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/13832

Glycerol standard
Add 10 µL of glycerol standard stock solution (1 mg/mL) into 990 µL 1X PBS buffer to generate 10 µg/mL standard solution (G7). Then perform 1:2 serial dilutions to get serially diluted glycerol standards (G6 - G1).

PREPARATION OF WORKING SOLUTION

Add 5 mL of Assay Buffer (Component C) into a bottle of Enzyme Mix (Component B) and mix well.

Add 25 µL of Amplite™ HRP substrate stock solution into the bottle of Component B + C and mix them well to make glycerol working solution (Component A + B + C). Note: This working solution is enough for one 96-well plate. It is not
stable, use it promptly. 

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of glycerol standards and test samples in a clear bottom 96-well microplate. G= Glycerol Standards (G1 - G7, 0.156 to 10 µg/mL), BL=Blank Control, TS=Test Samples.

BLBLTSTS
G1G1......
G2G2......
G3G3
G4G4
G5G5
G6G6
G7G7

Table 2. Reagent composition for each well.

WellVolumeReagent
G1 - G750 µLSerial Dilutions (0.156 to 10 µg/mL)
BL50 µL1X PBS Buffer
TS50 µLTest Sample
  1. Prepare glycerol standards (G), 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.
  2. Add 50 µL of glycerol working solution to each well of glycerol standard, blank control, and test samples to make the total glycerol assay volume of 100 µL/well. For a 384-well plate, add 25 µL of glycerol working solution into each well instead, for a total volume of 50 µL/well.
  3. Incubate the reaction at room temperature for 30 minutes to 1 hour, protected from light.
  4. Monitor the absorbance increase with an absorbance plate reader with path check on at OD of 575 nm.

Images


References


View all 47 references: Citation Explorer
Rapid monitoring of glycerol in fermentation growth media: Facilitating crude glycerol bioprocess development
Authors: Abad S, Perez X, Planas A, Turon X.
Journal: Talanta (2014): 210
Synthesis of substituted 1,3-diesters of glycerol using wittig chemistry
Authors: Lowe HI, Toyang NJ, Watson CT, Bryant J.
Journal: Nat Prod Commun (2014): 687
The glycoglycerolipid 1,2-dipalmitoyl-3-(N-palmitoyl-6'-amino-6'-deoxy-alpha-d-glucosyl)-sn-glycerol is no inhibitor of the human Myt1 kinase
Authors: Rohe A, Gollner C, Erdmann F, Sippl W, Schmidt M.
Journal: J Enzyme Inhib Med Chem (2014): 514
Functional characterization of Yersinia pestis aerobic glycerol metabolism
Authors: Willias SP, Chauhan S, Motin VL.
Journal: Microb Pathog (2014): 33
Quantitative analysis of glycerol levels in human urine by liquid chromatography-tandem mass spectrometry
Authors: Dong Y, Ma Y, Yan K, Shen L, Wang X, Xu Y, He G, Wu Y, Lu J, Yang Z, Feng F.
Journal: J Chromatogr B Analyt Technol Biomed Life Sci (2014): 30
Identification and glycerol-induced correction of misfolding mutations in the X-linked mental retardation gene CASK
Authors: LaConte LE, Chavan V, Mukherjee K.
Journal: PLoS One (2014): e88276
The MAPKK FgMkk1 of Fusarium graminearum regulates vegetative differentiation, multiple stress response, and virulence via the cell wall integrity and high-osmolarity glycerol signaling pathways
Authors: Yun Y, Liu Z, Zhang J, Shim WB, Chen Y, Ma Z.
Journal: Environ Microbiol (2014): 2023
An alcohol oxidase of Phanerochaete chrysosporium with a distinct glycerol oxidase activity
Authors: Linke D, Lehnert N, Nimtz M, Berger RG.
Journal: Enzyme Microb Technol (2014): 7
Bioelectrocatalytic sensor for triglycerides in human skin sebum based on enzymatic cascade reaction of lipase, glycerol kinase and glycerophosphate oxidase
Authors: Jeong CY, Han YD, Yoon JH, Yoon HC.
Journal: J Biotechnol (2014): 7
Isolation of peripheral blood mononuclear cells using glycerol density gradient
Authors: Aimola IA, Inuwa HM, Nok AJ, Mamman AI, Habila N, Muhammad A, Ndidi US, Ignatius B, J and e PL, Oghor R, Isaac LC, Afolabi-Balogun NB.
Journal: Cell Biochem Biophys (2014): 583