| Description | Free Fatty Acids (FFA), also known as Non-Esterified Fatty Acids (NEFA), are primarily produced by the hydrolysis of neutral fats. They are intermediate products in fat metabolism, involved in cell proliferation, inflammatory responses, and hormone regulation. FFA can also act as signaling moleculesFree Fatty Acids (FFA), also known as Non-Esterified Fatty Acids (NEFA), are primarily produced by the hydrolysis of neutral fats. They are intermediate products in fat metabolism, involved in cell proliferation, inflammatory responses, and hormone regulation. FFA can also act as signaling molecules with various physiological functions. Free fatty acids are closely related to lipid metabolism, glucose metabolism, and endocrine function. Their concentration is an important physiological and biochemical indicator, serving as an auxiliary parameter for disease evaluation and diagnosis, and also reflecting quality changes during food storage.Detection Principle: FFAs combine with copper ions to form copper soaps, which are soluble in chloroform. The copper ions can then react with a chromogenic solution to form a purplish-red complex. This product has a characteristic absorption peak at 550 nm. The FFA content can be quantified by measuring the change in absorbance.Detection Range: 0.0313 - 2 mMSensitivity: 0.0156 mMApplicable Samples: Serum (plasma), animal/plant tissues, cells, bacteria.A1492746Component48T96TStorageA1492746ACu Reagent6 mL12 mL2-8℃. Store in the dark.A1492746BChromogen15 mL30 mL2-8℃. Store in the dark.A1492746CStandard (16.41 mg Palmitic Acid)1 EA1 EA2-8℃. Store in the dark.User-Prepared Instruments and ReagentsMicroplate reader or visible spectrophotometer (capable of measuring absorbance at 550 nm)Incubator, Ice maker, Low-temperature centrifuge96-well plate or micro glass cuvettes, Adjustable pipettes and tipsHomogenizer (for tissue samples)Glass bottle (for preparing extraction buffer)n-Heptane, Anhydrous methanol, ChloroformExperimental Procedure1. Reagent PreparationReagent NameReagent PreparationPrecautionsExtraction Buffer (Self-prepared)In a glass bottle, mix Chloroform : n-Heptane : Anhydrous Methanol = 28 : 21 : 1. Cap tightly and mix well.Store at 4°C protected from light.Cu ReagentReady-to-use; equilibrate to room temperature before use; mix well before use.Store at 4°C protected from light.ChromogenReady-to-use; equilibrate to room temperature before use.Store at 4°C protected from light.StandardBefore use, dissolve contents in 1 mL of Extraction Buffer to obtain a 64 mM Standard solution. Mix well.Unused dissolved Standard can be stored in a tightly sealed glass bottle at 4°C protected from light for 1 month.2. Standard Curve SetupDilute the 64 mM Standard further with Extraction Buffer as shown in the table below.Standard No.Standard (µL)Extraction Buffer Volume (µL)Standard Concentration (mM)Std.120µL of 64mM stock6202Std.2100µL of Std.11001Std.3100µL of Std.21000.5Std.4100µL of Std.31000.25Std.5100µL of Std.41000.125Std.6100µL of Std.51000.0625Std.7100µL of Std.61000.0313Note: Prepare freshly diluted standards for each experiment.3. Sample PreparationNote: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 6 months.3.1 Animal Tissues: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 8,000 rpm, 4°C for 10 min. Collect the supernatant and keep on ice for detection.3.2 Plant Tissues: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and grind. Disrupt by ultrasonic homogenization on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 8,000 rpm, 4°C for 10 min. Collect the supernatant and keep on ice for detection.3.3 Cells or Bacteria: Collect 5 million cells or bacteria into a centrifuge tube. Wash with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer. Disrupt by ultrasonic homogenization on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 8,000 rpm, 4°C for 10 min. Collect the supernatant and keep on ice for detection.3.4 Serum (Plasma) and other liquids: Detect directly.4. Assay Steps4.1 Instrument Preparation: Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 550 nm. For spectrophotometers, zero the instrument with deionized water.4.2 Sample Assay (Add reagents sequentially to EP tubes):ReagentBlank Tube (µL)Standard Tube (µL)Test Tube (µL)Extraction Buffer240200200Various Std.0400Sample0040Cap the tubes tightly and vortex at medium speed for 30 seconds.Cu Reagent808080Cap the tubes tightly and vortex at medium speed for 30 seconds. Incubate at room temperature (25°C) for 20 minutes. Centrifuge at 2,000 g, room temperature (25°C) for 5 minutes.Upper Phase505050Chromogen2002002004.3 Incubate at room temperature (25°C) for 5 minutes. Transfer 200 µL from each tube to the corresponding wells of a 96-well plate or micro glass cuvettes. Measure the absorbance at 550 nm.Calculate ΔAtest=Atest-Ablank and ΔAstd=Astd-Ablank (The blank tube only needs to be set up once).Note: The measurement must be completed within 30 minutes after color development. It is recommended to perform preliminary experiments with 2-3 samples expected to have significant differences before formal testing. If Atest exceeds the detection range of the instrument, dilute the sample further with Extraction Buffer and multiply the result by the dilution factor.5. Result CalculationWe provide both derived and simplified calculation formulas, which are equivalent. The simplified formulas in bold are recommended as the final calculation formulas.5.1 Standard Curve PlottingPlot the standard curve with standard concentration as the y-axis and ΔAstd as the x-axis (using concentration as the y-axis facilitates calculation). Substitute ΔAtest into the standard curve equation to obtain y (mM).5.2 Sample FFA Content Calculation(1) Based on sample mass:FFA Content (µmol/g fresh weight) = y × Vextract ÷ W × n = y ÷ W × n(2) Based on bacterial or cell count:FFA Content (µmol/10⁴ cells) = y ÷ (Cell or Bacterial Count ÷ Vextract ) × n = y ÷ 500 × n = 0.002 × y × n(3) Based on liquid volume:FFA Content (µmol/L) = 1000 × y × nParameter Description:Vextract : Volume of Extraction Buffer added, 1 mLW: Sample mass, gn: Sample dilution factor (if further diluted)500: Cell or bacterial count, in units of 10⁴1000: Unit conversion factor, 1 L = 1000 mL6. Result PresentationTypical Standard Curve: y = 0.679x - 0.0109, R² = 0.9988(Free Fatty Acid (FFA) standard curve analyzed using a 96-well plate. Data and curve are for reference only; users must establish their own standard curve based on their experiment.)Precautions1. Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please implement appropriate biosafety precautions throughout the experiment. Wear personal protective equipment such as lab coats, masks, gloves, and hair caps. Perform experiments in a fume hood or biosafety cabinet.2. This product is for scientific research use only. Not intended for clinical diagnosis... Read More | Inquire | Inquire | Inquire | Product contentS665868Component50 TStorageS665868ABuffer GL25 mLRTS665868BBuffer GW1 (concentrate)13 mLRTS665868CBuffer GW2 (concentrate)15 mLRTS665868DBuffer GE15 mLRTS665868EProteinase K2×1.25 mLRTS665868FSpin Columns DM with Collection Tubes50 setsRTProduct IntroductionThis kit is suitable Product contentS665868Component50 TStorageS665868ABuffer GL25 mLRTS665868BBuffer GW1 (concentrate)13 mLRTS665868CBuffer GW2 (concentrate)15 mLRTS665868DBuffer GE15 mLRTS665868EProteinase K2×1.25 mLRTS665868FSpin Columns DM with Collection Tubes50 setsRTProduct IntroductionThis kit is suitable for the extraction of genomic DNA from fresh saliva or saliva/preservation solution mixture.The purification process of this product does not require the use of toxic solvents such as phenol or chloroform, and ethanol precipitation is not necessary. The optimized buffer system enables DNA to bind heterogeneously to the silica matrix centrifugal adsorption column, and the inhibitors of PCR and other enzymatic reactions can be effectively removed by a two-step washing step, and finally eluted with a low-salt buffer or water to obtain high-purity DNA.The purified obtained can be directly used for enzyme digestion, PCR, Real-Time PCR, library construction, Southern Blot, molecular labeling and other downstream experiments.Self-contained reagent: anhydrous ethanolPre-experiment Preparation and Important Notes1. Repeated freezing and thawing of the sample should be avoided, as this may result in smaller fragments of extracted DNA and a decrease in the amount extracted.2. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the label of the reagent bottle before first use.3. Before use, please check whether Buffer GL appears to be crystallized or precipitated.Redissolve in a 56°C water bath.4. If the downstream experiments are sensitive to RNA contamination, 4 µL DNase-Free RNase A can be added in step 3(100 mg/mL).5. For prolonged storage of salivary DNA at room temperature, our Salivary DNA Preservation Tubes are recommended.Operation steps1. Add 400 µL of saliva sample or saliva/preservation solution mixture.Note: 1) Saliva mixtures added to the preservation solution require a 50°C water bath for 1 hour or an empty 50°C temperature chamber for 2 hours prior to extraction.2) If an increase in sample volume is required, multiply the volumes of Proteinase K, Buffer GL, and anhydrous ethanol in Steps 2-4, and the liquid can be transferred in multiple times in Step 5.2. Add 40 µL of Proteinase K.3. Add 400µL Buffer GL, vortex and shake to mix thoroughly, and water bath at 56℃ for 15-30 minutes.Note: If RNA removal is required, add 4 µL of RNase A solution at a concentration of 100 mg/mL after the above steps are completed, vortex for 15 seconds, and leave at room temperature for 2 minutes.4. Centrifuge briefly to remove water droplets from the inside of the tube cap. Add 400 µL of anhydrous ethanol and mix well by vortexing and shaking. Centrifuge briefly.Note: 1) Vortex and shake to mix immediately after adding Buffer GL and anhydrous ethanol.The addition of Buffer GL and anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.2) A sol-gel product may be formed after GL and anhydrous ethanol, in which case vigorous shaking or vortexing is recommended.3) The solution obtained in the previous step is added to the adsorption column in the Collection Tube.5. (Spin Column DM) in the collection tube, and if the solution cannot be added all at once, it can be transferred in several times. centrifuge at 12,000 rpm (∼13,400 × g) for 1 min, pour off the waste solution in the collection tube, and put the adsorption column back into the collection tube.6. Add 500 µL of Buffer GW1 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.7. Add 500 µL of Buffer GW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.Note: Step 7 can be repeated if further DNA purity is required.8. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Leave the adsorption column at room temperature for several minutes to dry thoroughly.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).9. Place the adsorption column in a new centrifuge tube (supplied), add 50-200 µL of Buffer GE or sterilized water to the middle of the adsorption column overhanging the column, let it stand at room temperature for 2-5 minutes, and centrifuge at 12,000 rpm for 1 minute to collect the DNA solution.-20°C to preserve DNA.Note: 1) If the downstream experiment is sensitive to pH or EDTA, you can use sterilized water for elution. The pH of the eluent has a great influence on the elution efficiency, if water is used as the eluent should ensure that its pH is 7.0-8.5 (you can use NaOH to adjust the pH of the water to this range), and the elution efficiency is not high when the pH is lower than 7.0.2) Buffer GE preheated in a 65-70°C water bath and incubated at room temperature for 5 min before centrifugation can increase the yield.3) Because DNA preserved in water is subject to acidic hydrolysis, for long-term storage, elution with Buffer GE and storage at -20°C is recommended... Read More |