Invertase Assay Kit, BioAssay(TM)

Produktübersicht

• Artikelname: Invertase Assay Kit, BioAssay(TM)
• Artikelnummer: USB-I8447-02
• Hersteller Artikelnummer: I8447-02
• Alternativnummer: USB-I8447-02-100
• Hersteller: US Biological
• Kategorie: Kits/Assays

Produktbeschreibung

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: 1.5ml Assay Buffer: 10ml Enzyme Mix: 120ul Glucose Standard: 1ml Dye Reagent: 120ul Storage conditions: store 10x Reaction Buffer and Assay Buffer at 4C and other reagents at -20C. 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 100 100 2 60ul+40ul 100 60 3 30ul+70ul 100 30 4 0ul+100ul 100 0 Sample: 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. 30C). 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 as Invertase Activity = RSAMPLE-RCONTROL Slope 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 (Colorim