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Amplite® Fluorimetric Glycerol Assay Kit

Glycerol was measured with Amplite® Fluorimetric Glycerol Assay Kit on a solid black 96-well plate using a Gemini microplate reader.
Glycerol was measured with Amplite® Fluorimetric Glycerol Assay Kit on a solid black 96-well plate using a Gemini microplate reader.
Glycerol was measured with Amplite® Fluorimetric Glycerol Assay Kit on a solid black 96-well plate using a Gemini microplate reader.
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Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Glycerol is a precursor for the synthesis of triglycerides and phospholipids in 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® Fluorimetric Glycerol Assay Kit offers a sensitive fluorescence-based 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® HRP substrate in HRP-coupled reactions. The fluorescence signal can be measured by a fluorescence microplate reader. With this Fluorimetric Glycerol Assay Kit, we were able to detect as low as 0.015 mg/L (~0.16 µM) glycerol in a 100 µL reaction volume.


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black


Example protocol


Protocol summary

  1. Prepare Glycerol working solution (50 µL)
  2. Add Glycerol standards or test samples (50 µL)
  3. Incubate at room temperature for 10 - 30 minutes
  4. Monitor fluorescence increase at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important notes
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.


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. 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 Amplite™ HRP Substrate stock solution. Protect from light.

2. Glycerol standard solution (1 mg/mL):
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.


Glycerol standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13833

Add 10 μL of 1 mg/mL Glycerol standard solution into 990 μL 1x PBS buffer to generate 10 µg/mL Glycerol standard solution (GS7). Take 10 µg/mL Glycerol standard solution (GS7) and perform 1:3 serial dilutions in 1x PBS buffer to get serially diluted Glycerol standards (GS6-GS1).


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

2. Add 25 µL of 200X Amplite™ HRP Substrate stock solution into the bottle of Component B+C, and mix well to make Glycerol working solution. Note: This Glycerol working solution is enough for one 96-well plate. It is not stable, use it promptly.


Table 1. Layout of Glycerol standards and test samples in a solid black 96-well microplate. GS= Glycerol Standards (GS1 - GS7, 0.01 to 10 µg/mL), BL=Blank Control, TS=Test Samples. 


Table 2. Reagent composition for each well

GS1 - GS750 µLSerial Dilutions (0.01 to 10 µg/mL)
BL50 µL1X PBS Buffer 
TS50 µLtest sample
  1. Prepare Glycerol standards (GS), 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 10 - 30 minutes, protected from light.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm).



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