Fructose Assay Kit, BioAssay(TM)

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:. \n\nFructose Assay Kit reacts 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.\n\nKey Features:\nUse as little as 20ul samples. Linear detection range in 96-well plate: 12 to 1000uM fructose.\n\nApplications:\nDirect Assays: fructose in biological samples (e.g. serum, plasma, urine, saliva, milk, culture medium), food, juice, beverage and other agricultural products.\nDrug Discovery/Pharmacology: effects of drugs on fructose metabolism.\n\nKit Contents:\nAssay Buffer: 10ml Enzyme: 120ul\nPMS Solution: 1.5ml MTT Solution: 1.5ml\nStandard: 400ul 20mM D-Fructose\nStorage conditions. The kit is shipped on ice. Store all components at -20 degrees C. Shelf life of three months after receipt.\nPrecautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.\n\nAssay Procedure:\nNote: (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. 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 6 N HCl. Centrifuge 5 min at 14,000 rpm. Transfer 300ul supernatant into a clean tube and neutralize with 50ul 6 N NaOH. The neutralized supernatant is ready for assay (dilution factor n=1.36).\n1. Equilibrate all components to room temperature. Briefly centrifuge the tubes before opening. Keep thawed tubes on ice during assay.\n2. Standards: mix 12ul 20mM Standard with 228ul dH2O (final 1000uM). Dilute standard in dH2O as follows.\nNo 1000uM STD+H2O Vol (ul) Fructose (uM)\n1 100ul+0ul 100 1000\n2 60ul+40ul 100 600\n3 30ul+70ul 100 300\n4 0ul +100ul 100 0\nTransfer 20ul diluted standards into separate wells of a clear flatbottom 96-well plate.\nSamples: transfer 20ul of each sample into separate wells of the plate.\n3. Color reaction. Prepare enough Working Reagent by mixing, for each reaction well, 56ul Assay Buffer, 1ul 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.\n4. Read optical density at 565nm (520-600nm).\nNote: 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.\n\nCalculation:\nSubtract blank value (water, #4) from the standard values and plot the DOD against standard concentrations. Determine the slope and calculate the fructose concentration of Sample, [Fructose]=ODSample

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SPECIFICATIONS

Catalog Number

F8000-11

Size

1Kit

References

1. Novelli G, Reichardt JK. (2000). Molecular basis of disorders of human fructose metabolism: past, present, and future. Mol Genet Metab. 71: 62-65.\n2. Pudek MR et al. (1990). Low concentration fructose determination in plasma adapted to the Cobas-Bio. Clin Biochem. 23: 221-223.\n3. Gabrielli M. (1978). Serum fructose determination with centrifugal analyzers. Clin. Chem. 24: 1990-1995.