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Amplite® Colorimetric Enterokinase Activity Assay Kit
Example protocol
AT A GLANCE
Protocol summary
- Prepare test samples with diluted enterokinase standards (50 µL)
- Add equal volume of Enterokinase working solution (50 µL)
- Incubate at 37 °C for 30 - 60 minutes
- Monitor OD increase at 405 nm
Important notes
Thaw one vial of each kit component at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. EK Yellow™ stock solution (100X):
Add 50 µL of DMSO (Component E) into EK Yellow™ (Component A) to make 100X stock solution.
2. Enterokinase Substrate stock solution (100X):
Add 50 µL of DMSO (Component E) into Enterokinase Substrate (Component B) to make 100X stock solution.
3. Enterokinase standard solution (10 ug/mL):
Add 50 uL of ddH2O + 0.1% BSA into Enterokinase Standard vial (Component D) to make 10 µg/mL Enterokinase stock solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11410
Add 10 µL of 10 µg/mL Enterokinase standard solution into 990 µL of Assay Buffer (Component C) to get 100 ng/mL enterokinase solution (EK7). Then perform 1:2 serial dilutions in assay buffer to get serially diluted enterokinase standards (EK6 - EK1). Note: The EK standards are for positive control only, and should not be relied on as a quantitation standard for enzyme activity.
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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