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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 |