Amplite® Fluorimetric DPP4 Inhibitor Screening Kit

The Amplite® Fluorimetric DPP4 Inhibitor Screening Kit provides an efficient and direct method for evaluating potential DPP4 inhibitors. DPP4 activity is quantified by the enzymatic cleavage of a specific substrate, resulting in a fluorescent product (Ex/Em = 360/460 nm), which correlates directly with the enzymatic activity present. This kit includes Sitagliptin, a well-characterized DPP4 inhibitor used in diabetes treatment, as a reference control, facilitating the assessment of inhibitor efficacy. The assay is optimized for high-throughput screening applications. Dipeptidyl peptidase-4 (DPP4), also referred to as CD26, ADCP2, or DPP, is a transmembrane glycoprotein within the prolyl oligopeptidase family. DPP4 functions as a serine exopeptidase, cleaving N-terminal X-proline and X-alanine residues from polypeptides. It is implicated in numerous physiological processes, including glucose metabolism via regulation of glucagon-like peptide-1 (GLP-1), immune modulation through its role as a receptor on various immune cells, signal transduction as a transmembrane protein responsive to growth factors and chemokines, and tumor suppression via immune system interactions. Thus, DPP4 inhibitors are critical in the therapeutic management of type-2 diabetes.

Example protocol

AT A GLANCE

Important Note

Thaw all the kit components at room temperature before starting the experiment.

Protocol Summary

Prepare the test samples, and the serially diluted DPP4 Inhibitor (Sitagliptin) standards (50 μL).
Add the DPP4 working solution (50 μL).
Incubate at room temperature for 10-30 minutes.
Monitor the fluorescence intensity at Ex/Em=360/460 nm, cutoff=435 nm.

PREPARATION OF STANDARD SOLUTIONS

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/11324

DPP4 Inhibitor Standard

Add 2.5 μL of DPP4 Inhibitor (Sitagliptin) (Component D) to 497.5 µL of DPP4 Assay Buffer (Component A) to prepare a 50 μM DPP4 Inhibitor Sitagliptin solution (STD7). Then, take 250 μL of STD7 and perform 1:2 serial dilutions in DPP4 Assay Buffer (Component A) to create a series of Sitagliptin standards from STD7 to STD1.

PREPARATION OF WORKING SOLUTION

DPP4 Working Solution

Add 0.25 mL of the DPP4 Substrate (Component B) to 5 mL of the DPP4 Assay Buffer (Component A), and mix well.

Note: This DPP4 working solution should be freshly prepared before each experiment and protected from light. A 5 mL solution is enough for 100 tests. Please prepare the necessary amount of DPP4 working solution based on this proportion.

DPP4 Enzyme Working Solution

To prepare a 0.5 µg/mL DPP4 Enzyme solution, start with the DPP4 Assay Buffer (Component A). Add 5 µL of the DPP4 Positive Control Stock Solution to 995 µL of the DPP4 Assay Buffer. This mixture will result in a final concentration of 0.5 µg/mL DPP4 Enzyme.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of Sitagliptin standards and test samples in a 96-well solid black microplate. (STD = Sitagliptin Standards (STD7-STD1, 50 to 0.0781 µM), BL= Blank Control, TS = Test Samples.)

BL	BL	TS	TS
STD 1	STD 1	...	...
STD 2	STD 2	...	...
STD 3	STD 3
STD 4	STD 4
STD 5	STD 5
STD 6	STD 6
STD 7	STD 7

Table 2. Reagent composition for each well.

Well	Volume	Reagent
STD 1 -STD 7	50 µL	40 µL DPP4 Enzyme Solution + 10 µL Sitagliptin Serial Dilutions (0.078-50 @M)
BL	50 µL	DPP4 Assay buffer
DPP4 Enzyme	50 µL	40 µL DPP4 Enzyme Solution + 10 µL DPP4 Assay buffer
TS	50 µL	40 µL DPP4 Enzyme Solution + 10 µL Test Sample in DPP4 Assay Buffer

Prepare the Sitagliptin standards (STD1-7), blank controls (BL), DPP4 Enzyme, and test samples (TS) according to the layout provided in Tables 1 and 2. When using a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
Add 50 µL of DPP4 Working Solution to each well containing the blank control, DPP4 Enzyme, and test samples. For a 384-well plate, add 25 µL of DPP4 Working Solution to each well instead.
Incubate at room temperature for 10–30 minutes, protected from light.
Monitor the fluorescence intensity with a fluorescence microplate reader at Ex/Em = 360/460 nm, Cutoff = 435 nm.

References

View all 50 references: Citation Explorer

Comparison of incident hypertension between SGLT2 inhibitors vs. DPP4 inhibitors.
Authors: Suzuki, Yuta and Kaneko, Hidehiro and Okada, Akira and Komuro, Jin and Fujiu, Katsuhito and Takeda, Norifumi and Morita, Hiroyuki and Ako, Junya and Nishiyama, Akira and Yano, Yuichiro and Ieda, Masaki and Node, Koichi and Yasunaga, Hideo and Komuro, Issei
Journal: Hypertension research : official journal of the Japanese Society of Hypertension (2024)

Use of DPP4 Inhibitors and GLP-1 Receptor Agonists and Risk of Intestinal Obstruction: Scandinavian Cohort Study.
Authors: Ueda, Peter and Wintzell, Viktor and Melbye, Mads and Eliasson, Björn and Söderling, Jonas and Gudbjörnsdottir, Soffia and Hveem, Kristian and Jonasson, Christian and Svanström, Henrik and Hviid, Anders and Pasternak, Björn
Journal: Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Associatio (2024): 1226-1237.e14

Esophageal involvement in DPP4 inhibitor-associated bullous pemphigoid.
Authors: Brufau-Cochs, Magí and Alamon-Reig, Francesc and Luque-Luna, Mar and Bosch-Amate, Xavier and Moreta, Maria José and Bassegoda, Octavi and Mascaró, José M
Journal: Journal der Deutschen Dermatologischen Gesellschaft = Journal of the German Society of Dermatology : JDDG (2024): 430-432

Low dose sulphonylurea plus DPP4 inhibitor lower blood glucose and enhance beta cell function without hypoglycaemia.
Authors: Cordiner, Ruth Lorna Mary and Bedair, Khaled and Mari, Andrea and Pearson, Ewan
Journal: The Journal of clinical endocrinology and metabolism (2024)

Comparison of SGLT2 inhibitors vs. DPP4 inhibitors for patients with metabolic dysfunction associated fatty liver disease and diabetes mellitus.
Authors: Suzuki, Y and Kaneko, H and Okada, A and Ohno, R and Yokota, I and Fujiu, K and Jo, T and Takeda, N and Morita, H and Node, K and Yasunaga, H and Komuro, I
Journal: Journal of endocrinological investigation (2024): 1261-1270

Page updated on May 19, 2025

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