Free fatty acids, also known as non-esterified fatty acids, are aliphatic monocarboxylic acids that are ubiquitously found in animal or vegetable fat, oil and wax. This important lipid form, carried by albumin, is used as an energy source easily absorbed by muscles, brains, and other tissues and organs. Fatty Acids play important roles in cellular synthesis, energy metabolism and are implicated in diverse disorders such as diabetes mellitus, sudden infant death syndrome and Reye Syndrome.

The RayBiotech Non-Esterified Fatty Acids (NEFA/FFA) Detection Assay Kit provides a simple, reproducible, and sensitive tool for measuring free fatty acids in plasma, serum, cell lysates and other biological liquid samples. This assay uses a coupled enzymatic reaction system (ACS-ACOD Method), first, Acyl CoA Synthetase (ACS) catalyzes fatty acid acylation of coenzyme A. Next, the acyl-CoA product is oxidized by Acyl CoA Oxidase (ACOD), producing hydrogen peroxide which reacts with the Colorimetric Probe to form pink colored product. The optical density at 550nm is directly proportional to the free fatty acid concentration in the sample.

• Microplate reader capable of measuring absorbance at 550 nm
• Precision pipettes to deliver 2 µl to 1 ml volumes
• Distilled or deionized water
• Tubes to prepare sample dilutions
• Incubator at 37°C.

Each Fatty Acid standard and sample should be assayed in duplicate or triplicate. A freshly prepared standard curve should be used each time the assay is performed.

1. Add 20µL of each Fatty Acid standards and samples to each well.
2. Pipette 120ul reconstituted Fatty Acid Reagent A (see Reagent Preparation) to the 96-well microtiter plate. Gently shake the plate to ensure mixing.
3. Incubate for 5 minutes at 37°C.
4. Measure the absorbance (A1) of each well at 550nm.
5. Immediately, after the reading is over, add 40ul reconstituted Fatty Acid Reagent B (see Reagent Preparation) to each well. Gently shake the plate to ensure mixing. Ensure that there are no bubbles in the solution mixture.
6. Set a timer for 4 minutes at 37°C.
7. At the end of the 4 minutes read all wells at 550nm. Record all absorbencies (A2).