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An Optimized Firefly Luciferase Bioluminescent Assay for the Analysis of Free Fatty Acids
Author(s) -
Marques Simone M.,
Gonçalves Luís M.,
Esteves da Silva Joaquim C. G.
Publication year - 2015
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/php.12458
Subject(s) - luciferase , bioluminescence , chemistry , chromatography , luciferin , firefly protocol , detection limit , biochemistry , biology , zoology , transfection , gene
A firefly luciferase ( LUC )‐based bioluminescent assay for total free fatty acids ( FFA ) is presented. It is based on LUC 's capability of converting FFA into fatty acyl‐adenylates with consumption of adenosine 5′‐triphosphate ( ATP ). Since ATP is a cosubstrate in LUC 's bioluminescent reaction, together with firefly d ‐luciferin ( d ‐ LH 2 ) and atmospheric oxygen (O 2 ), any reduction in the assay's ATP content will lead to a decrease in the bioluminescent signal, which is proportional to the amount of FFA . Using FFA mixtures containing myristic (14:0), palmitic (16:0), stearic (18:0), oleic (18:1) and arachidonic acid (20:4) in ethanol, the assay was optimized through statistical experimental design methodology, namely fractional factorial (screening) and central composite (optimization) designs. The optimized method requires 2 μ L of sample per tube in a final reaction volume of 50 μ L. It is linear in the concentration range from 1 to 20 μ m , with limits of detection ( LOD ) and quantitation ( LOQ ) of 1.3 and 4.5 μ m , respectively. The method proved to be simple to perform, demands low reagent volumes, it is sensitive and robust and may be adapted to high‐throughput screening.