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Non-Esterified Free Fatty Acids (NEFA/FFA) Colorimetric Assay Kit– MSE Supplies LLC

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Non-Esterified Free Fatty Acids (NEFA/FFA) Colorimetric Assay Kit

SKU: E-BC-K013-M-96

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Non-Esterified Free Fatty Acids (NEFA/FFA) Colorimetric Assay Kit

SKU # E-BC-K013-M
Detection Instrument Microplate reader (690-730 nm,optimum wavelength: 715 nm)
Detection Method Colorimetric method


Product Details

Properties

Synonyms NEFA
Sample Type Animal tissue
Sensitivity 0.15 mmol/L
Detection Range 0.15-1.5 mmol/L
Detection Method Colorimetric method
Assay type Quantitative
Assay Time 30 min
Precision Average inter-assay CV: 5.100% | Average intra-assay CV: 3.300%
Other instruments required Vortex mixer, Micropipettor, Multichannel transferpettor (300 μL)
Storage 2-8℃
Valid period 12 months

 

Dilution Of Sample

It is recommended to take 2~3 samples with expected large difference to do pre-experiment before formal experiment and dilute the sample according to the result of the pre-experiment and the detection range (0.15-1.5 mmol/L).

The recommended dilution factor for different samples is as follows (for reference only):

Sample type Dilution factor
Rat liver tissue homogenate 1
Rat heart tissue homogenate 1
Rat kidney tissue homogenate 1
Mouse liver tissue homogenate 1

 

Note: The diluent is reagent 1.

 

Detection Principle

Under the condition of weak acidity, non-esterified free fatty acids (NEFA) react with nantokite to form copper soap, which has a specific absorption peak at 715nm. The content of NEFA can be calculated indirectly by measuring the OD value at 715 nm.


Kit Components & Storage

Item Component Size 1(48 T) Size 2(96 T) Storage
Reagent 1 Extracting Solution 60 mL ×1 vial 60 mL ×2 vials 2-8℃, 12 months
Reagent 2 10 mmol/L Palmitic Acid Standard 1 mL ×1 vial 1 mL × 2 vials 2-8℃, 12 months
Reagent 3 Control Solution 6 mL ×1 vial 12 mL × 1 vial 2-8℃, 12 months
Reagent 4 Reaction Solution 10 mL ×1 vial 20 mL × 1 vial 2-8℃, 12 months
Microplate 96 wells No requirement
Plate Sealer 2 pieces


Note: The reagents must be stored strictly according to the preservation conditions in the above table. The reagents in different kits cannot be mixed with each other. For a small volume of reagents, please centrifuge before use, so as not to obtain sufficient amount of reagents.

 

Technical Data:

Parameter:

Intra-assay Precision

Three rat liver samples were assayed in replicates of 20 to determine precision within an assay (CV = Coefficient of Variation).

Parameters Sample 1 Sample 2 Sample 3
Mean (mmol/L)
0.35 0.70 1.20
%CV 3.5 3.1 3.3

 

Inter-assay Precision

Three rat liver samples were assayed 20 times in duplicate by three operators to determine precision between assays.

Parameters Sample 1 Sample 2 Sample 3
Mean(mmol/L)
0.35 0.70 1.20
%CV 5.2 5.4 4.7

 

Recovery

Take three samples of high concentration, middle concentration and low concentration to test the samples of each concentration for 6 times parallelly to get the average recovery rate of 101%.


Standard 1 Standard 2 Standard 3
Expected Conc. (mmol/L) 0.35 0.8 1.1
Observed Conc. (mmol/L) 0.4 0.8 1.1
Recovery rate (%) 103 99 101

 

Sensitivity

The analytical sensitivity of the assay is 0.15 mmol/L NEFA. This was determined by adding two standard deviations to the mean O.D. obtained when the zero standard was assayed 20 times, and calculating the corresponding concentration.


Standard Curve:

As the OD value of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique or temperature effects), so the standard curve and data are provided as below for reference only:

Concentration (mmol/L) 0 0.3 0.4 0.6 0.9 1 1.2 1.5
Average OD 0.043 0.067 0.075 0.091 0.126 0.132 0.15 0.178
Absoluted OD 0 0.024 0.032 0.048 0.083 0.089 0.107 0.135