Amplite™ Fluorimetric Glycerol Assay Kit

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1e+410001001010.1- Dose-responseData legend Generated with Quest Graph™ Glycerol (ug/mL) RFU Hover mouse to interact
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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Unit Size: Cat No: Price (USD): Qty:
200 Tests 13833 $345


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

Ex/Em (nm)571/585
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsFluorescence microplate reader
Category Cell Biology
Cell Metabolism
Related Redox Enzymes
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 at Ex/Em= 540/590 nm. 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.




Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

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.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:540 nm
Emission:590 nm
Cutoff:570 nm
Recommended plate:Solid black
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. 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.

Preparation of 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).

Preparation of working solution

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.

Sample experimental protocol

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. 

BL BL TS TS
GS1 GS1 ... ...
GS2 GS2 ... ...
GS3 GS3    
GS4 GS4    
GS5 GS5    
GS6 GS6    
GS7 GS7    

Table 2. Reagent composition for each well

Well Volume Reagent
GS1 - GS7 50 µL Serial Dilutions (0.01 to 10 µg/mL)
BL 50 µL 1X PBS Buffer 
TS 50 µL test 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).
Example data analysis and figures

The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate Glycerol samples. We recommend using the Online Linear Regression Calculator which can be found at:

https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator

Figure 1. Glycerol was measured with Amplite™ Fluorimetric Glycerol Assay Kit on a solid black 96-well plate using a Gemini microplate reader.

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

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

Epigallocatechin-3-gallate (EGCG) inhibits 3-hydroxy-3-methylglutaryl-CoA reductase in the presence of glycerol
Authors: Wu Q, Li JZ, Zhao TY, Li TB, Wang JX, Sui Y.
Journal: Pak J Pharm Sci (2014): 1905

Functional characterization of Yersinia pestis aerobic glycerol metabolism
Authors: Willias SP, Chauhan S, Motin VL.
Journal: Microb Pathog (2014): 33

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

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, Jande PL, Oghor R, Isaac LC, Afolabi-Balogun NB.
Journal: Cell Biochem Biophys (2014): 583

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

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


View More Citations




Additional Documents

 
Safety Data Sheet (SDS)


Certificate of Analysis