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Amplite® Colorimetric Enterokinase Activity Assay Kit
Enterokinase (also called enteropeptidase) is a serine protease produced by cells in the duodenal wall and is a key enzyme in human and animal digestion system. Enterokinase converts trypsinogen into its active form trypsin, resulting in the subsequent activation of pancreatic digestive enzymes. The deficiency of enterokinase results in intestinal digestion impairment. The inhibition of enterokinase may have anti-tumor effects through suppressing proteases involved in carcinogenesis and metastasis. Therefore, highly selective and sensitive detection of enterokinase plays a key role in biochemical applications. Amplite® Colorimetric Enterokinase Activity Assay Kit offers a sensitive assay for quantifying enterokinase activity. After cleavage by enterokinase, the enterokinase substrate can be detected by EK Yellow™ in an absorbance microplate reader at 405 nm.
Example protocol
AT A GLANCE
- Prepare test samples and enterokinase standards (50 µL)
- Add equal volume of Enterokinase working solution (50 µL)
- Incubate at 37 °C for 30 - 60 minutes
- Monitor absorbance increase at 405 nm
Thaw one vial of each kit component at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTIONS
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
Add 50 µL of DMSO (Component E) into EK Yellow™ (Component A) to make 100X stock solution.
Add 50 µL of DMSO (Component E) into Enterokinase Substrate (Component B) to make 100X stock solution.
Add 50 µL of ddH2O + 0.1% BSA into Enterokinase Standard vial (Component D) to make 10 µg/mL Enterokinase stock solution.
PREPARATION OF STANDARD SOLUTIONS
https://www.aatbio.com/tools/serial-dilution/11410
PREPARATION OF WORKING SOLUTION
Add 50 µL of EK Yellow™ stock solution and 50 µL of Enterokinase Substrate stock solution into 5 mL of Assay Buffer (Component C); mix well to make Enterokinase (EK) working solution (Component A+B+C).
Note: The assay mixture is enough for one 96-well plate. It is not stable, use promptly.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of enterokinase standards and test samples in a 96-well clear bottom microplate. EK = enterokinase standard (EK1 - EK7, 1.56 to 100 ng/mL); BL = blank control; TS = test sample.
| BL | BL | TS | TS |
| EK1 | EK1 | ... | ... |
| EK2 | EK2 | ... | ... |
| EK3 | EK3 | ||
| EK4 | EK4 | ||
| EK5 | EK5 | ||
| EK6 | EK6 | ||
| EK7 | EK7 |
Table 2. Reagent composition for each well.
| Well | Volume | Reagent |
| EK1 - EK7 | 50 µL | serial dilution (1.56 to 100 ng/mL) |
| BL | 50 µL | Assay Buffer (Component C) |
| TS | 50 µL | sample |
- Prepare enterokinase standards (EK), blank controls (BL), and test samples (TS) into a 96-well clear bottom microplate according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
- Add 50 µL of EK working solution into each well of enterokinase standard, blank control, and test samples to make the total assay volume of 100 µL/well. For a 384-well plate, add 25 µL of EK working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction mixture at 37 °C for 30 - 60 minutes.
- Monitor the absorbance increase with an absorbance plate reader with path check on at OD of 405 nm.
References
Authors: Melicherova K, Krahulec J, Safranek M, Liskova V, Hopkova D, Szeliova D, Turna J.
Journal: Appl Microbiol Biotechnol. (2016)
Authors: Mbanefo EC, Kikuchi M, Huy NT, Shuaibu MN, Cherif MS, Yu C, Wakao M, Suda Y, Hirayama K.
Journal: PLoS Negl Trop Dis (2014): e2644
Authors: Liu Y, Ren L, Ge L, Cui Q, Cao X, Hou Y, Bai F, Bai G.
Journal: Biotechnol Lett (2014): 1675
Authors: Skala W, Goettig P, Br and stetter H., undefined
Journal: J Biotechnol (2013): 421
Authors: Chen Z, Han S, Cao Z, Wu Y, Zhuo R, Li W.
Journal: Peptides (2013): 145
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