Fructose (C6H12O6, also called levulose or laevulose), is a monosaccharide found in honey, tree fruits, berries, melons, and some root vegetables along with glucose and galactose. The human body can use fructose for energy, however, too much consumption may lead to high triglycerides.
Simple, direct and high-throughput assays for fructose determination find wide applications. This Fructose Assay Kit uses reagents that react directly and specifically with fructose to form a colored product. Glucose and galactose do not interfere. The color intensity at 565nm is directly proportional to the fructose concentration in the sample. As little as 20ul of sample can be used. The linear detection range (in 96-well plate) is 12-1000uM fructose.
Applications:Direct Assays: Fructose determination in biological samples (e.g. serum, plasma, urine, saliva, milk, culture medium), food, juice, beverage and other agricultural products
Drug Discovery/Pharmacology: Effects of drugs on fructose metabolism.
Storage and Stability:Store all kit components at -20C. Kit is stable for 6 months after receipt.
Precautions:This kit is for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents.
Procedural Notes:1. The following substances interfere and should be avoided in sample preparation: ascorbic acid, SDS (>0.2%), sodium azide, NP-40 (>1%) and Tween-20 (>1%). 2. This assay is based on a kinetic reaction. To ensure identical incubation time, addition of Working Reagent to standard and samples should be quick and mixing should be brief but thorough. Use of a multi-channel pipettor is recommended.
Reagent Preparation:Reconstitute Enzyme by adding 120ul Enzyme Buffer to the Enzyme tube. Make sure Enzyme is fully dissolved by pipetting up and down. Store reconstituted Enzyme at -20°C and use within 2 months.
Sample Treatment:Liquid samples such as serum, plasma and fruit juices can be assayed directly. Because fruit juices may contain high concentrations of fructose, it is recommended to dilute juice sample 50-fold (n = 50) in dH2O prior to assay. Milk samples should be cleared by mixing 600ul milk with 100ul 6N HCl. Centrifuge 5 min at 14,000rpm. Transfer 300ul supernatant into a clean tube and neutralize with 50ul 6N NaOH. The neutralized supernatant is ready for assay (dilution factor n = 1.36).
Assay Procedure:1. Equilibrate all components to room temperature. Briefly centrifuge the tubes before opening. Keep thawed tubes on ice during assay.
2. Standards: Mix 12ul 20mM Standard with 228ul dH2O (final concentration: 1000uM). Dilute standard in dH2O as follows:No1000uM STD + H2OVol (ul)Fructose (uM)1100ul + 0ul10010002 60ul + 40ul100 6003 30ul + 70ul100 3004 0ul + 100ul100 0
Transfer 20ul diluted standards into separate wells of a clear flat- bottom 96-well plate
Samples: Transfer 20ul of each sample into separate wells of the plate.
3. Color reaction. Prepare enough Working Reagent by mixing, for each reaction well, 56ul Assay Buffer, 1ul reconstituted Enzyme, 14ul PMS Solution and 14ul MTT Solution.
Keep Working Reagent protected from light. Add 80ul Working Reagent to each well. Tap plate to mix. Do not expose Working Reagent to light for more than 5 minutes. Incubate 60 min at room temperature in the dark.
4. Read optical density at 565nm (520-600nm)
Note: If the calculated fructose concentration of a sample is higher than 1000uM, dilute sample in water and repeat the assay. Multiply result by the dilution factor n.
Calculation:Subtract blank value (water, #4) from the standard values and plot the ΔOD against standard concentrations. Determine the slope and calculate the fructose concentration of Sample:Fructose concentration (uM) = [(ODSample - ODH2O)/ Slope (uM-1)] x n
where:ODSample and ODH2O are optical density values of the sample and water, respectively. n is the dilution factor
Conversions: 1mM fructose equals 18mg/dL, 0.018% or 180ppm.
