Free Fatty Acid Assay Kit (Amplex Red), IC-6572
Product Introduction
This product is a sensitive and specific fluorescence-based enzymatic assay kit for the quantitative measurement of free fatty acids (FFAs) in biological samples. Free fatty acids are critical metabolites involved in energy production, membrane biogenesis, and cellular signaling. Elevated FFA levels are associated with obesity, insulin resistance, type 2 diabetes, cardiovascular disease, and metabolic syndrome. This kit employs a coupled enzymatic reaction system in which free fatty acids are sequentially converted by acyl-CoA synthetase, acyl-CoA oxidase, and horseradish peroxidase (HRP) to generate the highly fluorescent product resorufin from the Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine). The fluorescence signal is directly proportional to the FFA concentration in the sample. The assay provides exceptional sensitivity, detecting sub-micromolar concentrations of free fatty acids, and is compatible with a wide range of biological samples including serum, plasma, cell culture supernatants, tissue homogenates, and lipid extracts.
Product Features
1. High Sensitivity: Amplex Red-based fluorometric detection enables quantification of FFA concentrations at sub-micromolar levels with excellent signal-to-noise ratios.
2. Specific Enzymatic Detection: Coupled enzyme cascade provides specific detection of free fatty acids with minimal interference from triglycerides, phospholipids, or other lipid species.
3. Broad Sample Compatibility: Validated for use with serum, plasma, cell culture media, tissue homogenates, and lipid extracts.
4. Convenient 96-Well Format: Compatible with high-throughput workflows and standard fluorescence microplate readers.
5. Complete Kit Format: Includes all necessary enzymes, cofactors, Amplex Red reagent, and palmitic acid standard for a complete assay workflow.
Specifications
Size: Available in 100 assays and 500 assays
Detection Method: Fluorescence (Ex/Em: 530-560 nm / 570-595 nm)
Format: 96-well plate
Sample Types: Serum, plasma, cell culture supernatants, tissue homogenates, lipid extracts
Detection Limit: Typically 0.1-1 µM (depending on sample type and preparation)
Storage and Stability
Storage Conditions: Store kit components at -20¡ãC, protected from light. The Amplex Red reagent is particularly light-sensitive and must be stored protected from light.
Shelf Life: The product is stable for 12 months from the date of manufacture when stored as directed. Avoid repeated freeze-thaw cycles. Reconstituted reagents should be used immediately or stored at -20¡ãC for up to 1 week.
Protocol (For Reference Only)
Important: Protect Amplex Red reagent and working solutions from light at all times. Use ultrapure water and clean glassware or plasticware to prevent fatty acid contamination. Prepare all working solutions freshly before use.
1. Sample Preparation: For serum or plasma samples, dilute samples 1:10 to 1:50 in assay buffer to bring FFA concentrations within the linear range of the standard curve. For cell culture supernatants, centrifuge at 1,000 x g for 5 minutes to remove debris before analysis. For tissue homogenates, homogenize tissue in cold PBS or assay buffer, centrifuge at 10,000 x g for 10 minutes at 4¡ãC, and collect the supernatant.
2. Standard Curve Preparation: Prepare a serial dilution of the provided palmitic acid standard in assay buffer to generate a standard curve typically covering a concentration range of 0-100 µM. Include a blank well containing assay buffer only.
3. Reaction Setup: Add 50 µL of each standard, diluted sample, and blank to designated wells of a black 96-well microplate with a clear flat bottom. Prepare Amplex Red Working Solution by mixing the provided components as directed in the detailed protocol, and add 50 µL to each well.
4. Incubation: Incubate the plate at 37¡ãC for 30 minutes protected from light. Monitor fluorescence development periodically; the reaction typically reaches completion within 30-60 minutes.
5. Detection and Analysis: Measure fluorescence using a microplate reader at excitation 530-560 nm and emission 570-595 nm. Subtract the blank value from all readings. Generate a standard curve by plotting fluorescence intensity versus standard concentration, and calculate FFA concentrations in unknown samples by interpolation.
Precautions
1. Amplex Red reagent is highly light-sensitive; prepare working solutions immediately before use and protect reaction plates from light during incubation.
2. Fatty acids are ubiquitous environmental contaminants; use clean glassware or plasticware, avoid handling with bare hands, and include appropriate blank controls in each assay.
3. Certain detergents, reducing agents, and metal ions may interfere with the assay; avoid contamination of reagents and samples with these substances.
4. The linear range of the assay may vary depending on the specific biological matrix; validate the linear range for each sample type by performing spike-recovery experiments.
5. For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: What free fatty acids are detected by this kit?
A1: The assay detects long-chain free fatty acids including palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and arachidonic acid (C20:4) with varying sensitivity. Short-chain fatty acids (C2-C6) are detected with lower efficiency. The provided palmitic acid standard serves as a representative fatty acid for quantification.
Q2: How should I dilute serum or plasma samples for this assay?
A2: Typical dilution factors range from 1:10 to 1:50 depending on the expected FFA concentration. Fasting human plasma FFA levels are typically 200-600 µM, requiring 1:20 to 1:50 dilution. Perform a preliminary experiment to determine the optimal dilution for your specific samples.
Q3: Can I measure free fatty acids in cell culture supernatants?
A3: Yes. Cell culture media containing serum or lipid supplements will have background FFA levels. Include appropriate medium-only controls and consider using serum-free or lipid-depleted media for metabolic studies. Centrifuge samples before analysis to remove any cellular debris.
Q4: What is the difference between this Amplex Red assay and colorimetric FFA kits?
A4: The Amplex Red-based fluorescence detection provides approximately 10- to 100-fold higher sensitivity compared to colorimetric methods, enabling detection of low micromolar FFA concentrations with smaller sample volumes. The fluorescence format also minimizes interference from colored or turbid samples.
Disclaimer
1. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
2. Due to the variable nature of biological research, optimization of sample preparation and dilution conditions is recommended for specific sample types and experimental systems.
3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages, including loss of samples or data.
4. Wear appropriate protective clothing and gloves when handling this product and biological samples.
Ordering Information
Catalog Number: IC-6572
Product Name: Free Fatty Acid Assay Kit (Amplex Red)
Size: 100 assays / 500 assays
Price:
100 assays: CNY ¥1950.00 / USD $195.00 / EUR €234.00 / JPY ¥35100.00
500 assays: CNY ¥4550.00 / USD $455.00 / EUR €546.00 / JPY ¥81900.00
2023 Version
