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Amplite® Fluorimetric DPP4 Activity Assay Kit
Example protocol
AT A GLANCE
Thaw all the kit components at room temperature before starting the experiment.
Prepare the test samples, DPP4 Positive Control, and the serially diluted AMC 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
https://www.aatbio.com/tools/serial-dilution/11323
PREPARATION OF 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.
Add 2.5 µL of DPP4 Inhibitor (Sitagliptin) (Component E) to 500 µL of DPP4 Assay Buffer (Component A) to create a 5X DPP4 Inhibitor Working Solution.
Prepare one or more DPP4 positive controls along with your test sample. The suggested concentration for these controls is between 0.5 µg/mL and 0.05 µg/mL in the DPP4 Assay Buffer (Component A). For example, to make a 0.5 µg/mL DPP4 positive control, add 2.5 µL of the DPP4 Positive Control Stock Solution to 250 µL of the DPP4 Assay Buffer (Component A).
Tissue and cells can be homogenized in the DPP4 Assay Buffer (Component A). After homogenization, centrifuge the sample at 13,000xg for 10 minutes to remove insoluble material. Serum samples can be directly added to the wells. Adjust the final volume of all samples to 50 µL using DPP4 Assay Buffer.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of AMC standards and test samples in a 96-well clear bottom microplate. (STD = AMC Standards (STD7-STD1, 10 to 0.0781 µM), BL= Blank Control, TS = Test Samples.)
BL | BL | Positive Control | Inhibitor Control | 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 | AMC Serial Dilutions (0.0781-10 µM) |
BL | 50 µL | DPP4 Assay buffer |
DPP4 Positive Control | 50 µL | 40 µL DPP4 Positive Control + 10 µL DPP4 Assay buffer |
DPP4 Inhibitor Control | 50 µL | 40 µL DPP4 Positive Control + 10 µL 5X DPP4 Inhibitor Working Solution |
TS | 50 µL | Cell content |
Prepare the AMC standards (STD1-7), blank controls (BL), DPP4 Positive Control, DPP4 Inhibitor Control, 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 Positive Control, DPP4 Inhibitor Control, 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
Authors: Preinfalk, Verena and Kimmeswenger, Isabella and Somoza, Veronika and Lieder, Barbara
Journal: Heliyon (2024): e30329
Authors: Mora-Rodríguez, José María and Sánchez, Belén G and Bort, Alicia and Díaz-Yuste, Alba and Ballester-González, Rubén and Arrieta, Francisco and Sebastián-Martín, Alba and Díaz-Laviada, Inés
Journal: Life sciences (2024): 122292
Authors: Wen, Xiaohui and Chang, Xiaona and He, Xueqing and Cai, Qingyun and Wang, Guang and Liu, Jia
Journal: The Journal of clinical endocrinology and metabolism (2024): 1517-1525
Authors: Martins, Flavia L and Ribeiro-Silva, Joao Carlos and Nistala, Ravi and Girardi, Adriana C C
Journal: American journal of physiology. Cell physiology (2024): C1203-C1211
Authors: Chahal, Sandhya and Rani, Payal and Shweta, and Goel, Kapil Kumar and Joshi, Gaurav and Singh, Rajvir and Kumar, Parvin and Singh, Devender and Sindhu, Jayant
Journal: Bioorganic chemistry (2024): 107363
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