May 12, 2026
Glycerol-P dehydrogenase enzyme assay
- 1Advanced Second Generation Biofuel (A2G) Laboratory, Universidade Estadual de Campinas (UNICAMP);
- 2Dartmouth College
- Unicamp
Protocol Citation: Isabela Uematsu Zambello, Daniel Olson 2026. Glycerol-P dehydrogenase enzyme assay. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp77b1gzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 12, 2026
Last Modified: May 12, 2026
Protocol Integer ID: 316913
Keywords: enzymatic assay for glycerol, simplified enzymatic assay, glycerol, enzyme
Funders Acknowledgements:
São Paulo Research Foundation (FAPESP)
Grant ID: 2021/05398-9
Abstract
Simplified enzymatic assay for Glycerol-P dehydrogenase activity.
Pre-incubation with TrisHCl Buffer
5m
Pre-incubate 10 µL of crude desalted cell extract with 90 µL of TrisHCl pH 7 Buffer for 5 minutes at 37°C.
Use an empty vector as a negative control.
5m
Tris HCl Buffer pH 7
- 100mM Tris HCl pH7
- 2mM DTT*
- 1mM NiCl2
*If using NiCl2, do not add DTT
Start reading the reaction at 340nm in a spectrophotometer.
Glycerol-P dehydrogenase reaction
30m
Add 0.2 mM NADH, mix by pipetting up and down.
- Suggested: 1 µL NADH 20 mM
In this step, the absorbance line should go up. Wait until it has stabilized.
Add 20 mM DHAP, mix by pipetting up and down.
- Suggested: 2 µL DHAP 1M
In this step, the absorbance line should go down.
Measure for 30 minutes or until the curve has stabilized.
Measure the slope of the curve and calculate the specific activity (ε = 6220 M−1 cm−1 for NADH)
30m