Overview

  • Product name

    Free Fatty Acid Assay Kit - Quantification
  • Detection method

    Colorimetric/Fluorometric
  • Sample type

    Cell culture supernatant, Urine, Serum, Plasma, Other biological fluids, Tissue Extracts
  • Assay type

    Quantitative
  • Sensitivity

    > 2 µM
  • Assay time

    0h 40m
  • Product overview

    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.


    In the free fatty acid assay protocol, fatty acids are converted to their CoA derivatives (coenzyme A), which are subsequently oxidized, leading to formation of color/ fluorescence. Fatty acids can then be easily quantified by either colorimetric (λ= 570 nm) or fluorometric (Ex/Em= 535/587 nm) methods. Palmitic acid is used to generate a standard curve.


    Free fatty acid assay protocol summary:
    - add samples and standards to wells
    - add reaction mix and incubate for 30 min at 37ºC
    - analyze with microplate reader

  • Notes

    Previously called Free Fatty Acid Quantification Assay Kit.

    This assay detects C-8 (octanoate) and longer fatty acids.

    Review our Metabolism Assay Guide to learn about assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also about how to assay metabolic function in live cells using your plate reader.
     

    How other researchers have used 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

  • Platform

    Microplate reader

Properties

Images

  • 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-?B pathway inhibitor, or vehicle (DMSO) for 48 hours prior to lipolysis assessment. ns, no significance; and ***P<0.001.

  • Plasma FFA levels was measured using ab65341 in male and female wild-type (WT) or AT2KO mice either on normal diet (ND) or high fat diet (HFD).

  • Colorimetric standard curve: mean of duplicates (+/-SD) with background readings subtracted.

  • Free Fatty Acid measured in biologicals showing concentration (µM)

  • Flourometric standard curve: mean of duplicates (+/-SD) with background readings subtracted.

Protocols

References

This product has been referenced in:

  • Becares N  et al. Impaired LXRa Phosphorylation Attenuates Progression of Fatty Liver Disease. Cell Rep 26:984-995.e6 (2019). Read more (PubMed: 30673619) »
  • Scopelliti A  et al. A Neuronal Relay Mediates a Nutrient Responsive Gut/Fat Body Axis Regulating Energy Homeostasis in Adult Drosophila. Cell Metab 29:269-284.e10 (2019). Read more (PubMed: 30344016) »
See all 77 Publications for this product

Customer reviews and Q&As

1-10 of 33 Q&A

Answer


Although we would suggest to use fresh tissue preferably, tissue that is snap-frozen in liquid nitrogen can be used too. The tissue can be ground and then lipid extraction can be done with choloroform-triton.

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Question
Answer

No, the kit does not detect triglycerides or other molecules that have a fatty acid components. The triglycerides need an enzyme to convert them to free fatty acids and this is not present in any of the kit components.

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Question
Answer

The reason heparinized plasma is not recommended for the free fatty acid assay is because heparin releases lipolytic enzymes that lead to an overestimation of FFA.

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Answer

You can test for endogoenous Acyl-CoAs in your samples by including sample wells without added ACS Reagent. In these wells, the long-chain free fatty acids will not be converted to coenzyme A, so if there are any interfering compounds including endogenous Acyl-CoAs, you can subtract this background from the +ACS Reagent wells.

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Question
Answer

We would recommend fresh serum samples for best results. If fresh samples is not an option storage at -80 °C is less likely to degrade the sample quality.

For this product ab65341, blood needs to be collected using an anticoagulant such as EDTA, sodium citrate, sodium fluoride, or ammonium oxalate. Heparinized plasma is not the best choice as heparin could interfere with the assay.

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Answer

Blood needs to be collected using an anticoagulant such as EDTA, sodium citrate, sodium fluoride, or ammonium oxalate. Heparinized plasma is not the best choice as heparin could interfere with the assay.

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Question
Answer

The details of the assay buffer is considered proprietary information. I can give you a pH range. It is between 7.5-8.2

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Question
Answer


This kit is good for C-8 or longer fatty acids so it should be suitable for measuring lipoxins.

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Question
Answer



Our scientists tried this assay with C7 and C6 fatty acids, but found that the final efficiency of detection decreases by ˜50% in that case.

Therefore I can confirm that the minimal length of suitable fatty acid is C8.

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Question
Answer

This kit will only detect the unbound free fatty acids in you sample and not any albumin bound free fatty acids,

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1-10 of 33 Q&A

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