Jun 19, 2026
DPP-IV inhibitory activity assay
- Marbio 1
- 1UiT The Arctic University of Norway
- Marbio
Protocol Citation: Marbio 2026. DPP-IV inhibitory activity assay. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lymd4elx9/v1
Manuscript citation:
https://link.springer.com/article/10.1007/s10811-013-0017-4
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: June 11, 2026
Last Modified: June 19, 2026
Protocol Integer ID: 318923
Keywords: DPP-IV inhibitory activity, Natural products, Biodiscovery, Bioprospecting, AMC (7-amino-4-methyl-coumarin), Diprotin A, insulinotropic potential in vivo, dpp4 gene, insulinotropic potential, major role in glucose metabolism, terminal dipeptides from peptide, iv inhibitory activity, type ii transmembrane glycoprotein, glucose metabolism, terminal dipeptide, peptide, inhibitory activity, dpp, observed dpp, enzyme, hydroxyproline
Abstract
DPP-IV, encoded by the DPP4 gene, is an enzyme expressed on the surface of most cell types. It is a type II transmembrane glycoprotein (a soluble form) and is present in blood plasma and various body fluids. DPP-IV preferentially cleaves N-terminal dipeptides from peptides with proline, alanine or hydroxyproline at the penultimate position. DPP-IV plays a major role in glucose metabolism. It is responsible for the degradation of incretins such as GLP-1, which results in the loss of their insulinotropic potential in vivo. This assay is to investigate whether the compounds are associated with the observed DPP-IV inhibitory activity. (reference: https://link.springer.com/article/10.1007/s10811-013-0017-4)
Materials
Samples:
Hydrolysates from cod, shrimp, salmon, and snow crab. Stock concentration is 10mg/ml.
Reagents
- Assay buffer: 0.02M Tris-HCl pH 8.0, 0.1M NaCl, 1mM EDTA.
- Gly-Pro-AMC HBr (7-amino-4-methyl-coumarin hydro bromide), Bachem, Product No: 4002520.0050
- DPP-IV, DPP (IV) human recombinant (Sigma:D3446-10UG).
- Diprotin A (Ile-Pro-Ile) Bachem, Product No: 4009723.0050.
- AMC (7-amino-4-methyl-coumarin), Bachem, Product No: 4001606.0001
Instruments:
- Black 96-well Immuno Plates Universal Binding (Thermo fisher scientific no: 9502867)
- Victor‱micro plate reader, 360nm excitation wave length and 460nm emission wave length.
Troubleshooting
Problem
DPP-V enzyme degraded
Solution
Contact the Sigma (using Cat no and Batch no) and ask for DPP-IV’s enzyme activity and concentration in detail. Such information can be huge different from the vial label. (https://www.sigmaaldrich.com/catalog/AdvancedSearchPage.do )
Safety warnings
Wear gloves during the procedure.
Before start
The hydrolysate samples should be prepared fresh or store the stock solution at -20°C.
Procedure
Sample preparation: Dilute (2-fold) the hydrolysate samples into assay buffer and shake for 30 minutes.
Load 10 µl of hydrolysates with 30 µl of assay buffer, 50 µl of 200 µM Gly-Pro-AMC and incubate for 5 min at 37°C.
Standard curve of AMC: (50 µM, 25 µM, 12.5 µM, 6.25 µM, and 0 µM) 50 µl of AMC into 50 µl of Assay buffer.
Measure the fluorescence of the samples by Victor microplate reader at excitation 360 nm and emission at 460 nm as T0’.
Add 10 µl of DPP-IV (0.8 Unit/µl) into the above mix and incubate for 30 min at 37°C.
Measure the fluorescence of the samples by Victor microplate reader at excitation 360 nm and emission at 460 nm as T30’.
IC50 inhibitory concentration that inhibits DPP-IV enzyme activity by 50%. The IC50 value reported for each hydrolysate sample is the mean value from three independent replicate assays.
Layout of 96 well plate
Notice:
Max reaction control: Using Assay buffer instead of hydrolysates. This indicates the full activity of DPP-IV. (40 µl of Assay buffer, 10 µl of DPP IV, and 50 µl GP-AMC)
Positive (inhibitory) control: Using Diprotin A as a reference DPP IV-inhibitory substance. (30 µl of Assay buffer, 10 µl of DPP IV, 10 µl of Diprotin A, and 50 µl GP-AMC)
If needed, background control: the fluorescence of the background. (50 µl of Assay buffer and 50 µl GP-AMC).
Protocol references
https://link.springer.com/article/10.1007/s10811-013-0017-4