Screen Quest™ Fluorimetric Neutrophil Elastase Inhibitor Screening Kit

Name: Screen Quest™ Fluorimetric Neutrophil Elastase Inhibitor Screening Kit
Brand: AAT Bioquest
SKU: 11802
Price: 595 USD
Availability: InStock

The Screen Quest™ Fluorimetric Neutrophil Elastase Inhibitor Screening Kit offers a fast, sensitive, and high-throughput method for screening and characterizing potential inhibitors of NE. This assay measures the NE activity based on hydrolysis of the substrate, producing a fluorescent product with Ex/Em=360/470nm. The amount of fluorescent product is directly proportional to the enzymatic activity of the neutrophil elastase present in the sample. Neutrophil elastase, also known as leukocyte elastase, ELANE, ELA2, elastase 2, neutrophil, elaszym, serine elastase, subtype human leukocyte elastase (HLE) is a cytotoxic Serine Protease with a broad substrate specificity. Azurophil granules of neutrophil store NE and release them in response to multiple stimuli like pathogens, immune complexes, or chemotactic agents such as PMA causing degradation of a range of extracellular matrix proteins, including fibronectin, laminin, proteoglycans, collagens, and elastin. NE hydrolysis accounts for approximately 80% of the total protease hydrolysis activity in the human body. Disease implications of NE include cystic fibrosis, COPD, lung emphysema, rheumatoid arthritis, and adult respiratory distress syndrome. NE has also been associated with lung injury seen in COVID-19.

Example protocol

AT A GLANCE

Protocol Summary

Prepare the NE enzyme solution (40 μL/well).
Prepare the NE inhibitor serial dilution, and add 10 μL/well.
Incubate the NE enzyme with inhibitor for 5-10 minutes at 37 °C.
Add 50 µL of the NE working solution.
Incubate at room temperature for 10-30 minutes.
Monitor fluorescence intensity at Ex/Em = 360/470 nm, Cutoff = 430 nm.

Important Note

Thaw all the kit components 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

NE Enzyme Stock Solution

Add 50 µL of the NE Assay Buffer (Component D) to the vial of NE Enzyme (Component B) to make a 100 µg/mL NE Enzyme stock solution. Mix well by pipetting, aliquot, and store at -80 °C.

Note: Must be used within 1 week of reconstitution. Avoid freeze/thaw cycles.

PREPARATION OF STANDARD SOLUTIONS

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/11802

NE Inhibitor Standard

Add 1.6 µL of NE Inhibitor (Component C) to 200 µL of NE Assay Buffer (Component D) to create an 80 µM NE Inhibitor solution (STD7). Take 100 µL of the STD7 solution and perform 1:2 serial dilutions using NE Assay Buffer (Component D). This process will produce a series of NE Inhibitor standards from STD7 to STD1.

PREPARATION OF WORKING SOLUTION

NE Enzyme Working Solution

Take 5 μL of the 100 μg/mL NE Enzyme stock solution and add it to 495 μL of NE Assay Buffer (Component D) to create a 1 μg/mL NE Enzyme solution.

Note: The concentration of the NE Enzyme solution needs to be optimized when using a different inhibitor.

NE Substrate Working Solution

Add 25 µL of NE Substrate (100X) (Component A) to 5 mL of NE Assay Buffer (Component D), and mix well.

Note: Prepare the NE working solution fresh before each experiment, and protect it from light.

Note: A 5 mL solution is sufficient for 100 tests. Adjust the amount of NE working solution proportionally to match the number of tests you need.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of NE inhibitor serial dilution and test samples in a 96-well solid black microplate. (STD = NE Inhibitor Standards (STD1-STD7, 1.25-80 µM), BL= Blank Control, TS = Test Samples.)

Positive Control	Positive Control	TS	TS
STD 1	STD 1	...	...
STD 2	STD 2	...	...
STD 3	STD 3	BL	BL
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 (Total 50 µL)
STD 1-STD 7	40 µL NE Enzyme Solution + 10 µL NE Inhibitor Serial Dilution
BL	40 µL NE Enzyme Solution + 10 µL NE Assay Buffer
GGT Positive Control	50 µL NE Assay Buffer
TS	40 µL NE Enzyme Solution + 10 µL of Inhibitor Test Sample

Prepare NE Inhibitor standards (STD1-7), blank controls (BL), positive control, and test samples (TS) as outlined in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
Incubate the NE enzyme with inhibitor for 5-10 minutes at 37 ⁰C.
Add 50 µL of NE Substrate Working Solution to each well containing the NE Inhibitor standards (STD1-STD7), blank control, positive control, and test samples. For a 384-well plate, add 25 µL of GGT Working Solution to each well instead.
Incubate for 10-30 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

View all 50 references: Citation Explorer

Neutrophil Elastase Remodels Mammary Tumors to Facilitate Lung Metastasis.
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

Human neutrophil elastase inhibitors: Classification, biological-synthetic sources and their relevance in related diseases.
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

Elevated first-trimester neutrophil elastase and proteinase 3 increase the risk of gestational diabetes mellitus and adverse fetal outcomes.
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

Neutrophil Elastase Degrades Histone Deacetylases and Sirtuin 1 in Primary Human Monocyte Derived Macrophages.
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)

Botulinum toxin type A ameliorates rat dorsal root ganglia neuron pyroptosis in postherpetic neuralgia by upregulating cathelicidin antimicrobial peptide to inhibit neutrophil elastase.
Authors: Wan, Quan
Journal: Chemical biology & drug design (2024): e14406

Page updated on September 5, 2024

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