Name: Free Fatty Acid Assay Kit - Quantification
SKU: ab65341
Rating: 5 (1 reviews)

Free Fatty Acid Assay Kit ab65341 is a quantitative, addition-only FFA assay with two 30 min incubations. In the assay, free fatty acids are converted to CoA derivatives, which are oxidized. Readout is on any colorimetric (570 nm) or fluorometric (Ex/Em 535/587 nm) plate reader.

- Used with sample types including plasma, serum, cell and tissue extracts, cell culture supernatants, and urine
- Cited in over 250 publications.
- Individual kit components also available for purchase with a minimum order of 20 units. Contact us to discuss your needs.

Images

Functional Studies - Free Fatty Acid Assay Kit - Quantification (AB65341), expandable thumbnail

Publications

Key facts

Detection method: Colorimetric/Fluorometric
Sample types: Cell Lysate, Urine, Plasma, Tissue Extracts, Cell culture supernatant, Serum, Other biological fluids
Assay type: Quantitative
Assay time: 60m
Sensitivity: > 2 µM

What's included?

2000 Test

Components

ACS Reagent

20 x 1 Vial

Acyl CoA Enzyme Mix

20 x 1 Vial

Assay Buffer V

20 x 25 mL

Enhancer I

20 x 200 µL

OxiRed Probe

20 x 200 µL

Palmitic Acid Standard

20 x 300 µL

Key facts

Detection method: Colorimetric/Fluorometric
Sample types: Cell Lysate, Urine, Plasma, Tissue Extracts, Cell culture supernatant, Serum, Other biological fluids
Assay type: Quantitative
Assay time: 60m
Assay Platform: Microplate reader
Sensitivity: > 2 µM

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage conditions: -20°C
Appropriate long-term storage conditions: -20°C
Storage information: -20°C

Notes

Free Fatty Acid Assay Kit ab65341 uses a convenient, sensitive enzyme-based method for detecting long-chain free fatty acids in various mammalian and other samples, such as serum, plasma and other body fluids, food, growth media, etc.

Other names used to refer to this type of assay include NEFA assay and FFA assay.

How the assay works
Free Fatty Acid Assay kit ab65341 measures non-esterified free fatty acids; specifically C-8 (octanoate) and longer fatty acids.

In the free fatty acid assay protocol, free fatty acids are converted to their coenzyme A derivatives by Acyl-CoA Synthetase. These are then oxidized by Acyl CoA Oxidase in a reaction that produces hydrogen peroxide. Hydrogen peroxide is then processed by an enzyme causing a reaction with a probe to generate red color (λmax = 570 nm) and fluorescence (Ex/Em = 535/587 nm). Palmitic acid is used to generate a standard curve.

The free fatty acid assay method using Acyl-CoA Synthetase and Acyl CoA Oxidase as used in ab65341 is the standard method used for free fatty acid assays.

Free fatty acid assay protocol summary

- Add samples and standards to wells.
- Add reaction mix and incubate for 30 min at 37°C.
- Analyze with microplate reader.

How other researchers are using Free Fatty Acid Assay Kit ab65341
This Free Fatty Acid assay kit has been used in publications in a variety of sample types, including:

- Human: cell culture lysates¹, serum²
- Mouse: plasma and liver tissue³, serum⁴, plasma⁵, heart tissue⁶, liver⁷
- Rat: serum⁸, plasma⁹, cytosol of articular chondrocyte primary cell cultures¹⁰
- Cell culture medium¹¹
- C reinhardtii algae¹²
- Drosophila¹³
- C elegans¹⁴

^{References: 1 - Ali A et al 2018, Phokrai P et al 2018; 2 - Tahapary DL et al 2018; 3 - Becares et al 2019; 4 - Pan J et al 2019, Maatta J et al 2018, Rohm M et al 2018, Shimazu T et al 2018; 5 - Woo M et al 2018, Tian X et al 2017; 6 - Liu W et al 2017; 7 - Hao et al 2017, Rui W et al 2016; 8 - Honore SM et al 2018; 9 - Van der Werf et al 2018;10 - Lee SW et al 2018; 11 - Kostrzewski T et al 2017; 12 - Ramanan R et al 2018; 13 - Scopelliti A et al 2019; 14 - Pollard AK et al 2019}

Related and recommended products
Free Fatty Acid assay kit ab65341is often used with Triglyceride assay kit Triglyceride Assay Kit - Quantification ab65336, and Cholesterol assay kit Cholesterol Assay Kit - HDL and LDL/VLDL ab65390, to study lipid metabolism and its role in various metabolic diseases such as diabetes, obesity, liver diseases, and cardiovascular conditions.

Other notes
This product is manufactured by BioVision, an Abcam company, and was previously called K612 Free Fatty Acid Quantification Assay Kit.

The Safety Datasheet for this product has been updated for certain countries. Please check the current version in the Support and downloads section.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

Free Fatty Acids (FFAs) also known as non-esterified fatty acids (NEFAs) are important in various biological processes. These molecules are derived from the hydrolysis of triglycerides and represent an important source of energy. They vary in mass depending on chain length and saturation; common examples include palmitic acid and oleic acid. FFAs exist extensively in plasma as they circulate bound to albumin reflecting their broad expression range across tissues. Researchers measure FFAs using FFA assay kits often employing colorimetric assay techniques in laboratory settings.

Biological function summary

FFAs play important roles in cellular energy metabolism by serving as substrates for beta-oxidation within mitochondria. They contribute to energy production especially in cardiac and skeletal muscle tissues. FFAs bind to mitochondrial membranes entering beta-oxidation pathways where they undergo sequential enzymatic processing. Although not typically part of large protein complexes FFAs interact with various enzymes and transport proteins facilitating metabolic processes.

Pathways

FFAs integrate into major metabolic processes such as the citric acid cycle and oxidative phosphorylation pivotal for ATP production. In lipid signalling pathways they serve as ligands for peroxisome proliferator-activated receptors (PPARs) influencing gene expression related to lipid metabolism. PPAR proteins including PPAR-alpha and PPAR-gamma are direct interactors within these pathways modulating lipid homeostasis and insulin sensitivity.

Associated diseases and disorders

Dysregulation of FFAs links to metabolic syndromes particularly obesity and type 2 diabetes. High levels of plasma FFAs contribute to insulin resistance and are associated with impaired glucose metabolism. In obesity excess FFAs activate inflammatory pathways leading to insulin receptor substrate (IRS) dysfunction. Additionally in type 2 diabetes FFAs impact the function of proteins like AMP-activated protein kinase (AMPK) disrupting glucose uptake and mitochondrial function exacerbating the disease phenotype.

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10 product images

Tian, Xiaoxiang et al., PloS one vol. 12,5 e0176873., Fig 6, doi:10.1371/journal.pone.0176873
Functional Studies - Free Fatty Acid Assay Kit - Quantification (ab65341)
Lipolysis were evaluated by measuring free fatty acids released into the culture medium of siCreg1 or si-Scramble 3T3-L1 cells using ab65341, which were treated with BMS-345541 (5 μmol/L), an NF-κB pathway inhibitor, or vehicle (DMSO) for 48 hours prior to lipolysis assessment. ns, no significance; and ***P<0.001.
Samuel P et al., PLoS One 8(1), Fig 7a. DOI: 10.1371/journal.pone.0048425. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/
Functional Studies - Free Fatty Acid Assay Kit - Quantification (ab65341)
Plasma FFA levels were measured using ab65341 in male and female wild-type (WT) or AT₂KO mice either on normal diet (ND) or high fat diet (HFD).
Functional Studies - Free Fatty Acid Assay Kit - Quantification (ab65341)
Func S-1.
Colorimetric standard curve: mean of duplicates (+/-SD) with background readings subtracted.
Functional Studies - Free Fatty Acid Assay Kit - Quantification (ab65341)
Free Fatty Acid measured in biologicals showing concentration (μM).
Functional Studies - Free Fatty Acid Assay Kit - Quantification (ab65341)
Flourometric standard curve: mean of duplicates (+/-SD) with background readings subtracted.
Biochemical assay - Free Fatty Acid Assay Kit - Quantification (ab65341)
Triglyceride assay kit Triglyceride Assay Kit - Quantification ab65336, Cholesterol assay kit Cholesterol Assay Kit - HDL and LDL/VLDL ab65390, and Free Fatty Acid assay kit ab65341 used with rat serum and rat liver.
Li et al. used Triglyceride assay kit Triglyceride Assay Kit - Quantification ab65336, Cholesterol assay kit Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation ab65359, and Free Fatty Acid assay kit ab65341 to investigate the impact of using L. reuteri treatment to ameliorate the impact of biorhythm disorder-ignited dyslipidemia in long-term darkness treated rats.
The concentrations of triglycerides (TG), total cholesterol (CHOL), HDL-cholesterol (HDL-C), and free fatty acids (NEFA) in rat sera and liver tissues were detected using Triglyceride Quantification Assay Kit (Triglyceride Assay Kit - Quantification ab65336), HDL and LDL/VLDL Cholesterol Assay Kit (Cholesterol Assay Kit - HDL and LDL/VLDL ab65390). and Free Fatty Acid Quantification Assay Kit (ab65341), respectively. All procedures were carried out following the recommended instructions provided by the kit manufacturers.
Biochemical assay - Free Fatty Acid Assay Kit - Quantification (ab65341)
Diagram showing the principles of the Free Fatty Acid assay method.
Biochemical assay - Free Fatty Acid Assay Kit - Quantification (ab65341)
Triglyceride assay kit Triglyceride Assay Kit - Quantification ab65336 and free fatty acid assay kit ab65341 used with liver tissue and plasma samples respectively.
Samuel et al. used the triglyceride assay kit and free fatty acid assay kit to understand gender differences in metabolic syndrome disorders in angiotensin receptor AT2R knockout mice.
Plasma free fatty acid levels were measured by colorimetric method using the Abcam free fatty acid quantification kit.
Hepatic triglyceride levels was measured using Triglyceride Assay Kit - Quantification ab65336 in male and female wild-type (WT) or AT2KO (knockout) mice with either normal diet (ND) or high fat diet (HFD). For hepatic triglyceride measurement, livers were dissected and snap-frozen in liquid nitrogen and stored at −80°C. In brief, lipids were extracted by homogenizing 25 mg liver tissue in 1 ml 5% Triton-X100 in water, then slowly heated to 80°C in water bath for 5 min. The samples were cooled down and again heated to solubilize all triglycerides into solution. The samples were centrifuged for 5 min and supernatants were diluted 10 fold with dH2O for quantification.
Biochemical assay - Free Fatty Acid Assay Kit - Quantification (ab65341)
Triglyceride assay kit Triglyceride Assay Kit - Quantification ab65336 and free fatty acid assay kit ab65341 used with heart tissue.
Han et al. used Triglyceride assay kit Triglyceride Assay Kit - Quantification ab65336 and Free Fatty Acid assay kit ab65341 to investigate cardiac abnormalities in high fat diet fed mice when researching the role of the long non-coding RNA Lipid-Droplet Transporter (LIPTER).
The concentration of free fatty acids (FA) (f, n = 5 mice each group) were quantified using the Free Fatty Acid Assay kit (ab65341), and triglyceride (TG) concentrations (g, n = 7 mice each group) were quantified using the Triglyceride Quantification Assay Kit (Triglyceride Assay Kit - Quantification ab65336).
Approximately 25 mg of mouse heart tissue per heart was collected and homogenized in 500 μl lipid extraction buffer (Lipid Extraction Kit (Chloroform Free) ab211044) using a Beadbug Homogenizer. After centrifugation, the supernatant was removed to a new tube. FA or TAG concentration was measured according to the manufacturer’s protocols. The FA and TAG concentrations in WT and genetically modified mouse hearts were normalized to the collected heart tissue weights.
Biochemical assay - Free Fatty Acid Assay Kit - Quantification (ab65341)
Triglyceride assay kit Triglyceride Assay Kit - Quantification ab65336 and Free Fatty Acid assay kit ab65341 used with mouse liver tissue.
Yue et al. used triglyceride assay kit Triglyceride Assay Kit - Quantification ab65336 and free fatty acid assay kit ab65341 to investigate the protective effects of IL-22 on alleviating alcoholic hepatitis.
Administration of IL-22 reduces alcohol-induced lipid accumulation in mouse liver. PF (pair fed), AF (alcohol fed), AF+IL-22 (alcohol fed with IL-22 treatment).
(A) BODIPY 493/503 staining of mouse liver. Neutral lipids were stained in green, and nuclei were counterstained in blue. (B) Hepatic triglyceride (TG) and free fatty acid (FFA) levels.
Quantification assays of triglycerides (TG) and free fatty acids (FFA) in the liver were conducted using commercial kits from Abcam (Waltham, MA; Triglyceride Assay Kit - Quantification ab65336 and ab65341) per the manufacturer’s instructions.