Urease Assay Kit, BioAssay(TM)

Produktübersicht

• Artikelname: Urease Assay Kit, BioAssay(TM)
• Artikelnummer: USB-U2015-02
• Hersteller Artikelnummer: U2015-02
• Alternativnummer: USB-U2015-02-96
• Hersteller: US Biological
• Kategorie: Kits/Assays

Produktbeschreibung

Urease (Amidohydrolase, EC 3.5.1.5) is an enzyme that catalyzes the hydrolysis of urea into carbon dioxide and ammonia. (NH2)2CO+H2O CO2+2NH3 Many gastrointestinal or urinary tract pathogens produce urease. Thus its activity is a useful diagnostic parameter for the presence of pathogens such as Helicobacter pylori. Urease is found in bacteria, yeast, and higher plants. Urease activity is commonly determined in anaerobes of the bovine rumen, human feces and environmental samples such as soils and phytoplanktons. Urease Assay Kit provides a very sensitive and convenient means to measure urease activity in a variety of samples including soil. In the assay, urease reacts with urea, resulting in the formation of ammonia, which is determined by the Berthelot method at 670nm. The assay is simple, sensitive, stable and high-throughput adaptable. Key Features: Safe. Non-radioactive assay. Sensitive and accurate. As low as 0.003 U/L urease 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: Urease activity determination in biological and environmental samples. Evaluation and screening for urease inhibitors. Kit Contents: Assay Buffer: 20ml (pH 7.0) Reagent A: 12ml Urea: 1.5ml Reagent B: 6ml NH4Cl: 100ul 50mM Storage conditions: store all reagent at 4C. This product is shipped at room temperature. Shelf life of 6 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: ammonia is known to interfere with this assay and prior to assay, should be removed by dialysis or filtration. 1. Assay Preparation. Prior to assay, bring all components to room temperature. For calibration curve, prepare a 500uM premix by mixing 5ul 50mM NH4Cl and 495ul Buffer. Dilute NH4Cl as follows No Premix+Buffer Vol (ul) NH4 + (uM) 1 100ul+0ul 100 500 2 80ul+20ul 100 400 3 60ul+40ul 100 300 4 40ul+60ul 100 200 5 30ul+70ul 100 150 6 20ul+80ul 100 100 7 10ul+90ul 100 50 8 0ul+100ul 100 0 Transfer 90ul into separate wells of a clear flat-bottom 96-well plate. Samples. Dilute sample in Assay Buffer (Note: it is prudent to test different dilutions to ensure urease activity is within the detection range). Transfer 90ul sample into separate wells. Use 90ul enzyme buffer as a Sample Blank. 2. Enzyme Reaction. Add 10ul Urea to each well. Incubate at desired temperature for 10 min. 3. Detection. Add 100ul Reagent A to each well. Tap plate to mix. Then add 50ul Reagent B to each well. Tap plate to mix again. Note: addition of Reagent A terminates the urease reaction. Incubate for 30 min in the dark. Read optical intensity at 670nm (630-700nm). Calculation: Plot NH4Cl calibration curve and determine its Slope (uM-1). Urease enzyme activity in the sample is calculated as Urease Activity = ODSAMPLE-ODBLANK Slope x t (U/L) where ODSAMPLE and ODBLANK are the measured OD values of the Sample and Sample Blank (enzyme buffer). t is the incubation time (10 min) for standard urease assay. If urease activity is higher than 25 U/L, dilute enzyme in assay buffer, repeat assay and multiply the calculated activity by the dilution factor. Unit definition: one unit of urease catalyzes the formation of 1uMole ammonia per min at pH 7.0 under the assay conditions. General Considerations: (1). Soil and other environmental samples can be extracted in Assay Buffer (10mM sodium phosphate, pH 7.0) using any established methods. For such low urease activity samples, incubate the urease reaction for 2 to 4 hours at 30 or 37C (Step 2). Soil samples may contain very low concentrations of ammonia. To correct for sample ammonia, immediately prior to detection (Step 3), prepare