Amplite® Fluorimetric Lipoxygenase Inhibitor Screening Kit Green Fluorescence

Product key features

High sensitivity and specificity: Accurately measures the inhibition of lipoxygenase activity in samples.
Convenient: Ready-to-use reagents.
Versatility: Suitable for various sample types such as cell lysates, purified enzymes, or tissue samples.
Rapid detection: Obtain results in less than 30 minutes.

Product description

The Amplite® Fluorimetric Lipoxygenase Inhibitor Screening Kit offers a robust, quick and convenient method for screening and evaluating compounds that inhibit lipoxygenase (LOX) activity in biological samples. Lipoxygenases are iron-containing oxidoreductase enzymes that catalyze the dioxygenation of unsaturated fatty acids, such as arachidonic acid and linoleic acid, to produce hydroperoxides. These hydroperoxides act as intermediates in the production of leukotrienes and lipoxins, which are signaling molecules involved in inflammatory responses, cancer, and other biological processes.

Our Amplite® Lipoxygenase Inhibitor Screening Kit utilizes a fluorescence detection system to monitor the inhibition of lipoxygenase activity. The substrate is oxidized by lipoxygenase in the sample, producing a fluorogenic compound that generates a fluorescence signal (Ex/Em = 490/530 nm). The presence of an inhibitor reduces the enzymatic activity, leading to a decrease in the fluorescence signal. This assay provides a simple, reliable solution for identifying potential inhibitors of lipoxygenase.

This kit is compatible with fluorescence microplate readers and is ideal for high-throughput screening of inhibitors, studying enzyme kinetics, or investigating disease-related pathways involving lipoxygenase.

Example protocol

AT A GLANCE

Protocol summary

Prepare LOX enzyme solution (40 μL)
Prepare inhibitor serial dilution and add 10 μL
Incubate enzyme with inhibitor for 5-10 minutes at RT
Add LOX working solution (50 μL)
Incubate 10-30 min at RT
Monitor fluorescence intensity at Ex/Em = 490 nm/ 530 nm (Cutoff= 515 nm)

Important notes
Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STANDARD SOLUTIONS

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/15252

LOX Inhibitor Standard Solution:

Add 8 μL of LOX Inhibitor (Component E) to 992 µL PBS to get 200 µM of LOX inhibitor (Std 7). Take 500 µL (Std 7) and perform 1:2 serial dilutions in PBS to get serially diluted standards (Std 6-Std 1).

PREPARATION OF WORKING SOLUTION

LOX Enzyme (Lipooxygenase Enzyme):

Add 50 μL PBS to LOX Enzyme (Component C) to prepare 1 mg/ mL of stock solution. Mix well by pipetting up and down. Store at < -15 ⁰C. Avoid repeated freeze/thaw cycles.

Add 4 µL of 1 mg/ml LOX to 996 µL PBS+0.1% BSA (not included) to get 4 µg/ml LOX enzyme solution.

LOX Working Solution

Add 20 µL of Arachidonic Acid (Component D), 50 µL LOX Green (Component A) into 5 mL of LOX Assay Buffer (Component B), and mix well to make a LOX working solution.

Note: This LOX working solution should be prepared freshly before the experiment, and kept from light. 5 mL is for 100 tests; please prepare the amount of LOX working solution as needed proportionally.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1: Layout of LOX inhibitor standards and test samples in a solid black 96-well microplate. STD = LOX inhibitor Standards (STD 1-STD 7, 0.6 to 40 µM), BL=Blank Control, TS=Test Samples.

BL	BL	TS	TS
STD1	STD1	...	...
STD2	STD2	...	...
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 (total 50 μL)
STD1-STD7	40 μL LOX enzyme dilution+10 μL LOX inhibitor serial dilution
BL Control	PBS+0.1% BSA: 50 μL
TS	LOX enzyme solution 40 μL+10 μL test inhibitor sample
Positive control	LOX enzyme solution 40 μL+10 μL PBS

1. Prepare LOX inhibitor standards (STD1-7), blank controls (BL), LOX positive control and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
2. Incubate LOX enzyme solution with LOX inhibitor solution for 10 min at RT.
3. Add 50 µL of LOX Working Solution to each well of standard, blank control, positive control and test samples. For a 384-well plate, add 25 µL of LOX Working Solution into each well instead.
4. Incubate at 10–20 minutes at RT, protected from light.
5. Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em= 490 nm/ 530 nm (Cutoff = 515 nm).

References

View all 50 references: Citation Explorer

Bio-affinity ultrafiltration combined with UPLC Q-Exactive Plus Orbitrap HRMS to screen potential COX-2 and 5-LOX inhibitors in mulberry (Morus alba L.) leaf.
Authors: Du, Yan and Chen, Yu-Lang and Zhang, Ying and Zhao, Yan-Lin and Huang, Zhong and Jin, Peng and Ji, Shuai and Tang, Dao-Quan
Journal: Journal of ethnopharmacology (2025): 119325

Exploring new imines as anti-inflammatory COX and 5-LOX inhibitors with an improved pharmacokinetic profile.
Authors: Said, Mona F and Moussa, Bahia A and Mahrouse, Marianne A and Marie, Sarah M and Mohamed, Nada M
Journal: Future medicinal chemistry (2024): 311-334

Probing N-substituted 4-(5-mercapto-4-ethyl-4H-1,2,4-triazol-3-yl)-N-phenylpiperdine-1-carboxamides as potent 15-LOX inhibitors supported with ADME, DFT calculations and molecular docking studies.
Authors: Nawaz, Zahid and Riaz, Naheed and Saleem, Muhammad and Iqbal, Ambar and Ejaz, Syeda Abida and Muzaffar, Saima and Bashir, Bushra and Ashraf, Muhammad and Rehman, Aziz-Ur and Bilal, Muhammad Sajjad and Prabhala, Bala Krishna and Sajid, Salvia
Journal: Heliyon (2024): e35278

In Vitro Evaluation of Pharmacokinetic Properties of Selected Dual COX-2 and 5-LOX Inhibitors.
Authors: Bošković, Jelena and Dobričić, Vladimir and Savić, Jelena and Rupar, Jelena and Aleksić, Mara and Marković, Bojan and Čudina, Olivera
Journal: Pharmaceuticals (Basel, Switzerland) (2024)

Discovery of new thymol-3,4-disubstituted thiazole hybrids as dual COX-2/5-LOX inhibitors with in vivo proof.
Authors: El-Miligy, Mostafa M M and Al-Kubeisi, Ahmed K and Nassra, Rasha A and El-Zemity, Saad R and Hazzaa, Aly A
Journal: Journal of enzyme inhibition and medicinal chemistry (2024): 2309171

Page updated on February 12, 2025

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