Amplite® Fluorimetric Lipooxygenase Activity Assay kit Green Fluorescence

Product key features

High sensitivity and specificity: Detects low levels of lipoxygenases 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 Lipooxygenase Activity Assay kit offers a robust, convenient and rapid method for measuring lipoxygenase (LOX) activity in biological samples. Lipoxygenases are iron-containing oxidoreductase enzymes that are involved in the dioxygenation of unsaturated fatty acids, such as arachidonic acid and linoleic acid, to produce hydroperoxides. These hydroperoxides serve as intermediates for the production of leukotrienes and lipoxins, which are signaling molecules that regulate inflammatory responses involved in inflammation, cancer, and other biological processes.

Our Amplite® Fluorimetric Lipooxygenase Activity Assay kit detects lipoxygenases based on a fluorescence detection system. The substrate is oxidized by lipoxygenase present in the sample, producing a fluorigenic compound resulting in an enhanced fluorescence signal with Ex/Em=490/530nm. The generated signal is directly proportional to the enzymatic activity, enabling accurate quantification of the enzyme. This kit provides a simple, reliable solution for detecting lipoxygenase activity in various biological samples, including cell lysates, serum, purified enzymes, and tissue homogenates. It is compatible with fluorescence microplate readers and is ideal for studying enzyme kinetics, screening inhibitors, or investigating disease-related pathways.

Example protocol

AT A GLANCE

Protocol summary

1. Prepare test samples along with LOX positive control and Rhodamine standard (50 μL)
2. Add LOX working solution (50 μL)
3. Incubate for10–30 minutes at RT
4. 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/15251

Rhodamine 123 Standard

Add 2 μL of Rhodamine 123 Standard (Component E) to 998 µL PBS+0.1% BSA (not included) to get 10 µM of Rhodamine 123 standard (Std7). Take 500 µL (Std7) and perform 1:2 serial dilutions in PBS+0.1% BSA to get serially diluted standards (Std 6-Std 1).

PREPARATION OF WORKING SOLUTION

Lipooxygenase Positive Control (LOX Positive Control)

Add 50 μL PBS to LOX Positive Control (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 enzyme to 996 µL PBS+0.1% BSA to get 4 µg/ml LOX enzyme. LOX working solution is not stable and should be used promptly.

LOX Working Solution

Add 20 µL of Arachidonic Acid (Component D) into 5.0 mL of LOX Assay Buffer (Component B), and then add 50 µL of LOX Green (Component A) and mix well to make LOX working solution.

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

SAMPLE EXPERIMENTAL PROTOCOL

Table 1: Layout of Rhodamine 123 standards and test samples in a solid black 96-well microplate. STD = Rhodamine123 Standards (STD 1-STD 7, 0.15 to 10 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	Reagent
STD1-STD7	50 µL	Serial Dilutions (0.15 to 10 µM)
BL Control	50 µL	PBS+0.1% BSA
LOX Positive Control	50 µL	LOX positive control 4 µg/ml
TS	50 µL	LOX Sample

1. Prepare Rhodamine123 standards (STD1-7), Blank Controls (BL), LOX Positive Control and LOX 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. Add 50 µL of LOX Working Solution to each well of standard, blank control, LOX positive control and test samples. For a 384-well plate, add 25 µL of LOX working solution into each well instead.
3. Immediately after addition of the LOX Working Solution, measure the fluorescence (RFU) using the preset plate reader settings (Ex = 490 nm/Em = 530 nm, Cutoff=515nm) in kinetic mode reading every 30 seconds for a total of 20 minutes at room temperature. The standard curve can be read together with the sample or at the end of the incubation time.

References

View all 50 references: Citation Explorer

Seasonal metabolic variation in the essential oil composition of various Tabebuia species and evaluation of their anti-inflammatory activity in vitro and in silico.
Authors: Khaled, Nesma and Ibrahim, Nehal and Youssef, Fadia S and El-Ahmady, Sherweit H
Journal: Archiv der Pharmazie (2025): e2400710

The Potential Therapeutic Approach of Ursodeoxycholic Acid as a Potent Activator of ACE-2 on Cerebral Disorders Induced by γ-irradiation in Rats.
Authors: Galal, Shereen Mohamed and El Kiki, Shereen Mohamed and Elgazzar, Eman Mahmoud
Journal: Cell biochemistry and function (2024): e70024

12/15-Lipooxygenase Inhibition Reduces Microvessel Constriction and Microthrombi after Subarachnoid Hemorrhage in Mice.
Authors: Dienel, Ari and Hong, Sung Ha and Zeineddine, Hussein A and Thomas, Sithara and Shafeeque, C M and Jose, Dania A and Torres, Kiara and Guzman, Jose and Dunn, Andrew and P Kumar, T and Rao, Gadiparthi N and Blackburn, Spiros L and McBride, Devin W
Journal: Research square (2024)

12/15-Lipooxygenase Inhibition Reduces Microvessel Constriction and Microthrombi After Subarachnoid Hemorrhage in Mice.
Authors: Dienel, Ari and Hong, Sung Ha and Zeineddine, Hussein A and Thomas, Sithara and M, Shafeeque C and Jose, Dania A and Torres, Kiara and Guzman, Jose and Dunn, Andrew and T, P Kumar and Rao, Gadiparthi N and Blackburn, Spiros L and McBride, Devin W
Journal: Translational stroke research (2024)

Design, Synthesis, Biological Evaluation, and Molecular Docking Study of 4,6-Dimethyl-5-aryl/alkyl-2-[2-hydroxy-3-(4-substituted-1-piperazinyl)propyl]pyrrolo[3,4-c]pyrrole-1,3(2H,5H)-diones as Anti-Inflammatory Agents with Dual Inhibition of COX and LOX.
Authors: Redzicka, Aleksandra and Wiatrak, Benita and Jęśkowiak-Kossakowska, Izabela and Kochel, Andrzej and Płaczek, Remigiusz and Czyżnikowska, Żaneta
Journal: Pharmaceuticals (Basel, Switzerland) (2023)

Page updated on February 12, 2025

Ordering information

Price
Unit size
Catalog Number	15251
Quantity

Add to cart

Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Technical Support	Contact us
Purchase order	Send to sales@aatbio.com
Shipping	Standard overnight for United States, inquire for international