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
Invertase Assay Kit provides a convenient and ultra-sensitive colorimetric and fluorometric 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 fluorometric method (lem/ex=585/530nm). The assay is simple, sensitive, stable and high-throughput adaptable.
Key Features:Safe. Non-radioactive assay
Sensitive and accurate. As low as 0.007 U/L invertase activity can be quantified
Homogeneous and convenient. "Mix-incubate-measure" type assay
No wash and reagent transfer steps are involved
Robust and amenable to HTS: can be readily automated on HTS liquid handling systems for processing thousands of samples per day.
Applications:Invertase and sucrase activity determination in biological and environmental (e.g. soil) samples. Evaluation and screening for invertase inhibitors.
Kit Contents:10x Reaction Buffer: 12ml (pH 4.5) 10x Sucrose: 1.5mlAssay Buffer: 10ml Enzyme Mix: 120ulGlucose Standard: 1ml Dye Reagent: 120ulStorage conditions: store 10x Reaction Buffer and Assay Buffer at 4°C and other reagents at -20°C. This product is shipped on ice. Shelf life of 3 months after receipt
Precautions: 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.
Assay Procedure:Interference: 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
1. 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
No 100uM Std+H2O Vol (ul) Glucose (uM)1 100ul+0ul 100 1002 60ul+40ul 100 603 30ul+70ul 100 304 0ul+100ul 100 0Sample: transfer 40ul sample to separate wells of the plate. As a sample control, use 40ul diluted Reaction Buffer
2. Enzyme Reaction. Add 5ul of the diluted Sucrose to each well. Tap plate to mix. Incubate 20 min at desired temperature (e.g. 30°C)
3. 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 Fluorometric 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.
Calculation:Plot glucose standard curve and determine its Slope (uM-1). Invertase enzyme activity in the sample is calculated asInvertase Activity =RSAMPLE–RCONTROLSlope x t(U/L)where RSAMPLE and RCONTROL are the OD or fluorescence values of the sample and sample control (i.e. Reaction Buffer). t is the incubation time (20 min)
Unit definition: one unit of invertase catalyzes the formation of 1uMole glucose per min at pH 4.5 under the assay conditions
Note: if the OD or fluorescence intensity is higher than the value for 100uM glucose (Colorimetric Assay:) or 20uM (Fluorometric Assay:), dilute sample in 1-fold Reaction Buffer and repeat the assay. Multiply the result by the enzyme dilution factor.
Invertase Assay In Soil Samples:Soil samples can be directly assayed as follows. Weigh about 100 mg soil into a 1.5-mL Eppendorf tube. Add 880ul diluted Reaction Buffer and 120ul diluted sucrose. Mix thoroughly by homogenization and/or vortexing. Immediately remove 200ul mixture into a clean tube and centrifuge for 2 min at 14,000 rpm. Transfer 100ul clear supernatant into another clean tube and immediately freeze at -20°C. This "time zero" sample serves as a sample control. Incubate the invertase reaction for 1 hour at 30 or 37°C (Step 2). Centrifuge for 2 min at 14,000 rpm. Transfer 40ul clear supernatant and the above sample control for glucose determination (Step 3).
Materials Required, But Not Provided:Pipetting devices, centrifuge tubes, clear or black flat bottom 96-well plate (e.g. Corning Costar).
