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Amplite® Fluorimetric Neutrophil Elastase Activity Assay Kit
Example protocol
AT A GLANCE
Thaw all the kit components at room temperature before starting the experiment.
Prepare the test samples, and the serially diluted NE standards (50 μL).
Add the NE working solution (50 μL).
Incubate for 10-20 minutes at 37 °C.
Monitor the fluorescence intensity at Ex/Em=360/470 nm, cutoff=430 nm.
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 10 μL of NE Dilution Buffer (Component C) to prepare a 100 μg/mL stock solution. Mix thoroughly by pipetting up and down.
Note: Store the stock solution at -80 ⁰C, avoid repeated freeze/thaw cycles.
PREPARATION OF STANDARD SOLUTIONS
https://www.aatbio.com/tools/serial-dilution/11327
PREPARATION OF WORKING SOLUTION
To prepare the NE working solution, add 25 µL of NE Substrate (100X) (Component A) to 5 mL of NE Assay Buffer (Component D) and mix thoroughly.
Note: Prepare the NE working solution fresh before each experiment and protect it from light. A 5 mL solution is sufficient for 100 tests. Prepare the NE working solution in the required amount, adjusted proportionally to the number of tests needed.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of NE standards and test samples in a 96-well solid black microplate. (STD = NE Standards (STD1-STD7, 4.1 to 1000 ng/mL), 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 | Serial Dilutions (4.1 to 1000 ng/mL) |
BL | 50 µL | NE Dilution Buffer (Component C) |
TS | 50 µL | Test Sample |
Prepare the NE standards (STD1-STD7), blank controls (BL), and test samples (TS) as outlined in Tables 1 and 2. When using a 384-well plate, add 25 µL of reagent per well instead of 50 µL.
Add 50 µL of NE Working Solution to each well for NE standards, blank controls, and test samples. For a 384-well plate, add 25 µL of NE Working Solution to each well.
Incubate for 10-20 minutes at 37 °C, protected from light.
Monitor the fluorescence intensity with a fluorescence microplate reader at Ex/Em= 360/470 nm (Cutoff = 430 nm).
References
Authors: Lulla, Amriti R and Akli, Said and Karakas, Cansu and Caruso, Joseph A and Warma, Lucas D and Fowlkes, Natalie W and Rao, Xiayu and Wang, Jing and Hunt, Kelly K and Watowich, Stephanie S and Keyomarsi, Khandan
Journal: Molecular cancer therapeutics (2024): 492-506
Authors: Ocampo-Gallego, Jhonnatan Styver and Pedroza-Escobar, David and Caicedo-Ortega, Ana Ruth and Berumen-Murra, María Teresa and Novelo-Aguirre, Ana Lucía and de Sotelo-León, Rebeca Denis and Delgadillo-Guzmán, Dealmy
Journal: Fundamental & clinical pharmacology (2024): 13-32
Authors: Wang, Lihong and Zhou, Zhoujunhao and Xu, Xinming and Li, Yue and Zhang, Rui and Yu, Zhiyan and Huang, Xinmei and Zang, Shufei and Sun, Tiange
Journal: Reproductive biology and endocrinology : RB&E (2024): 2
Authors: Zheng, Shuo and Bulut, Gamze B and Kummarapurugu, Apparao B and Ma, Jonathan and Voynow, Judith A
Journal: International journal of molecular sciences (2024)
Authors: Wan, Quan
Journal: Chemical biology & drug design (2024): e14406
Safety data sheet