Free Fatty Acid Assay Kit (Fluorometric)

Produktnummer ABIN5067612

Produktdetails

Nachweismethode: Fluorometric

Applikation: Biochemical Assay (BCA)

Proben: Plasma, Serum

Sensitivität: 5 μM

Produktmerkmale: Free Fatty Acid Assay Kit measures non-esterified fatty acids (NEFA) in serum and plasma by 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 kit's Fluorometric Probe (Ex. 530-560 nm/Em. 585-595 nm). The Free Fatty Acid Assay Kit is a simple, fluorometric assay that quantitatively measures the free fatty acid concentration (non-esterified) in various samples using a 96-well microtiter plate format. Each kit provides sufficient reagents to perform up to 100 assays, including blanks, standards and unknown samples. The kit contains a palmitic acid standard and has a detection sensitivity limit of ~5 μM. Note: This kit is not suitable for urine or heparin-containing samples. Fatty acids (C8 and longer) can be quantified with this kit.

Bestandteile:

1. FFA Standard : One 100 μL vial of 50 mM palmitic acid in ethanol.
2. 20X Assay Buffer : One 1.5 mL vial.
3. 5X Enzyme Mixture A : Four 1 mL vials (containing ACS, Ascorbate Oxidase, and necessary cofactors).
4. 5X Enzyme Mixture B : Four 0.5 mL vials (containing ACOD).
5. NEM Reagent : One 150 μL amber vial.
6. Fluorometric Probe : One 110 μL amber vial.

Anwendungsinformationen

Applikationshinweise: Optimal working dilution should be determined by the investigator.

Kommentare:

• Measures non-esterified fatty acids (NEFA) in serum or plasma
• Available with colorimetric or fluorometric detection
• Standard included for quantitative results

Aufbereitung der Reagenzien:

• FFA Standard: Thaw at 37 °C for 10 minutes. Once homogeneous and mixed well, maintain the standard at room temperature during assay preparation. The solution is stable for 1 week at room temperature. For longer term storage, the solution should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws.
• 1X Assay Buffer: 20X Assay Buffer should be thawed/maintained at 4 °C during assay preparation. Dilute the 20X Assay Buffer with deionized water. Stir to homogeneity. The 1X solution is stable for 1 week at 4 °C. For longer term storage, any unused 20X stock material should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws.
• 1X Enzyme Mixture (A or B): Each 5X Enzyme Mixture should be thawed/maintained at 4 °C during assay preparation. Dilute the 5X Enzyme Mixture with cold, deionized water. Stir to homogeneity. The 1X solution is stable for 1 week at 4 °C. For longer term storage, any unused 5X stock material should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws. Note: These components are provided in multiple tubes to minimize multiple freeze/thaws.
• NEM Reagent: Thaw and maintain at 4 °C during assay preparation. The solution is stable for 1 week at 4 °C. For longer term storage, the solution should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws.
• Fluorometric Probe: Thaw and maintain at room temperature during assay preparation. Any unused material should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws.

Aufbereitung der Proben:

• Urine: This kit is not recommended for urine samples.
• Plasma: Collect blood with an anticoagulant such as citrate, EDTA or oxalate and mix by inversion. Centrifuge the blood at 1000 x g at 4 °C for 10 minutes. Collect plasma supernatant without disturbing the white buffy layer. Sample should be tested immediately or frozen at -80 °C for storage. Plasma must be diluted before assaying (1:2 to 1:20 in 1X Assay Buffer). Normal FFA levels in human plasma are typically 150-450 μM. Note: Heparin is known to interfere with the assay. Heparin containing samples including heparinized plasma should be avoided.
• Serum: Collect blood in a tube with no anticoagulant. Allow the blood to clot at room temperature for 30 minutes. Centrifuge at 2500 x g for 20 minutes. Remove the yellow serum supernatant without disturbing the white buffy layer. Samples should be tested immediately or frozen at -80 °C for storage. Serum must be diluted before assaying (1:2 to 1:20 in 1X Assay Buffer). Normal FFA levels in human serum are typically 100-700 μM.

Testdurchführung:

Each FFA 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 10 μL of the FFA standards, samples or blanks to the 96-well microtiter plate.
2. Add 200 μL of 1X Enzyme Mixture A (see Preparation of Reagents) to each well.
3. Cover the plate wells to protect the reaction from light.
4. Incubate at 37 °C for 30 minutes.
5. During the step 4 incubation, separately prepare the desired volume of Detection Enzyme Mixture according to Table 2 below, based on the number of tests to be performed. Maintaining all components and mixtures at 4 °C throughout this step, add components in the following sequence: a. In a tube, add the appropriate volume of 1X Enzyme Mixture B (see Preparation of Reagents). b. To the 1X Enzyme Mixture B add the corresponding volume of NEM Reagent. Mix well. c. Finally, add the corresponding volume of Fluorometric Probe. Mix well and immediately use. Note: Detection Enzyme Mixture will appear slightly pink in color. This is normal background and should be subtracted from all absorbance values. 1X Enzyme NEM Reagent Fluorometric Total Volume of # of Tests in Mixture B (μL) Probe (μL) Detection 96-well Plate (mL) Enzyme Mixture (100 μL/test) (mL) 10 100 100 10.2 100 5 50 50 5.1 50 2.5 25 25 2.55 25 Table
6. Preparation of Detection Enzyme Mixture 5
7. Transfer 100 μL of the above Detection Enzyme Mixture to each well (from step 4).
8. Cover the plate wells to protect the reaction from light.
9. Incubate at 37 °C for 10 minutes.
10. Read the plate with a fluorescence microplate reader equipped for excitation in the 530-560 nm range and for emission in the 585-595 nm range.
11. Calculate the concentration of free fatty acid within samples by comparing the sample fluorescence to the standard curve. Negative controls (without FFA) should be subtracted.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: -80 °C

Informationen zur Lagerung: Store the entire kit at -80°C. Avoid multiple freeze/thaws by aliquoting. The NEM Reagent and Fluorometric Probe are light sensitive and should be maintained in amber tubes.

Antigendetails

Hintergrund: Triglycerides (TAG) are a type of lipid in the blood, serving as an energy source and playing a key role in metabolism. Triglycerides are the digestive end product of breaking down dietary fats. Any extra carbohydrates and fats that are not immediately used are chemically converted into triglycerides. In the intestines, secreted enzyme lipases hydrolyse the triglyceride ester bond, yielding glycerol and free fatty acids (FFA) in a process called lipolysis. Additionally, hormones induce and regulate lipase activity in adipose tissue, resulting in changes to blood FFA levels. Free fatty acids then bind plasma albumin for circulation in the body, serving as a readily absorbed energy source for muscle, brain and other organ tissues. Measurement of free fatty acids has become useful in monitoring and diagnosis of several diseases and metabolic disorders (e.g. obesity, insulin resistance, diabetes, cancer).