Invertase Assay Kit, BioAssay(TM)

Invertase (b-fructofuranosidase, EC 3.2.1.26) is an enzyme that catalyzes the hydrolysis of sucrose to fructose and glucose. Invertases cleave at the O-C(fructose) bond, whereas a related enzyme sucrase (EC 3.2.1.48) cleaves at the O-C(glucose) bond. A wide range of microorganisms produce invertase and can, thus, utilize sucrose as a nutrient. Invertase assay finds wide applications in environmental (e.g. soil), agricultural and food (confectionery) industry.\nInvertase Assay Kit provides a convenient and ultra-sensitive colorimetric and fluorimetric means to measure invertase activity. In the assay, invertase cleaves sucrose, resulting in the formation of fructose and glucose, which is determined by a colorimetric (570nm) or fluorimetric method (lem/ex=585/530nm). The assay is simple, sensitive, stable and high-throughput adaptable.\n\nKey Features:\nSafe. Non-radioactive assay.\nSensitive and accurate. As low as 0.007 U/L invertase activity can be quantified.\nHomogeneous and convenient. "Mix-incubate-measure" type assay.\nNo wash and reagent transfer steps are involved.\nRobust and amenable to HTS: can be readily automated on HTS liquid handling systems for processing thousands of samples per day.\n\nApplications:\nInvertase and sucrase activity determination in biological and environmental (e.g. soil) samples. Evaluation and screening for invertase inhibitors.\n\nKit Contents:\n10x Reaction Buffer: 12ml (pH 4.5) 10x Sucrose: 1.5ml\nAssay Buffer: 10ml Enzyme Mix: 120ul\nGlucose Standard: 1ml Dye Reagent: 120ul\nStorage conditions: store 10x Reaction Buffer and Assay Buffer at 4 degrees C and other reagents at -20 degrees C. This product is shipped on ice. Shelf life of 3 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:\nInterference: thiols (b-mercaptoethanol, dithioerythritol etc) at > 10uM interfere with this assay and should be avoided. Glucose, if present in the sample, should be removed by dialysis or membrane filtration.\n1. Assay Preparation. Prior to assay, bring all components to room temperature, briefly centrifuge tubes before opening. Dilute the provided 10x Reaction Buffer and 10x Sucrose to 1-fold by mixing 1 vol of the reagent with 9 vol of dH2O. Use the diluted reagents for all assays. For glucose standard curve, mix 5ul Glucose Standard with 828ul dH2O (final 100uM). Dilute as follows and transfer 40ul standards to separate wells in a clear flat-bottom 96-well plate.\nNo 100uM Std+H2O Vol (ul) Glucose (uM)\n1 100ul+0ul 100 100\n2 60ul+40ul 100 60\n3 30ul+70ul 100 30\n4 0ul+100ul 100 0\nSample: transfer 40ul sample to separate wells of the plate. As a sample control, use 40ul diluted Reaction Buffer.\n2. Enzyme Reaction. Add 5ul of the diluted Sucrose to each well. Tap plate to mix. Incubate 20 min at desired temperature (e.g. 30 degrees C).\n3. Glucose Determination. Prepare enough Working Reagent in bulk. For each well, mix 95ul Assay Buffer, 1ul Enzyme Mix, 1ul Dye Reagent. Add 90ul Working Reagent to each well. Immediately tap plate to mix. Incubate for 20 min in the dark. Read OD570nm. Note: the procedure for Fluorimetric Assay:s is the same except that (1) a black flat-bottom 96-well plate is used, (2) glucose standards should be at 20, 12, 6 and 0uM and that fluorescence intensity at lem/ex=585/530nm is measured.\n\nCalculation:\nPlot glucose standard curve and determine its Slope (uM-1). Invertase enzyme activity in the sample is calculated as\nInvertase Activity =\nRSAMPLE

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