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Production of Urea occurs in the liver and it is secreted from the kidney. Toxic ammonia is removed from our body in the form of urea. Kidney function of a patient is assessed by determination of level of urea. If urea level is increased, it will cause obstruction in urinary tract, nephritis, renal ischemia and results in diabetes, congestive heart failure, liver diseases. On the other hand if urea level is decreased it will result in severe hepatic insufficiency. Measurement of concentration of urea in biological samples followed by direct, simple and ready automation procedures are very important in the discovery of new Drugs and its research work. Urea Assay Kit of Bioassay Systems is designed in such a manner so that concentration of urea in biological samples can be measured directly without any previous treatment. In Urea Assay Kit method chromogen is used that on reaction with urea results in the formation of the Colored complex and the color intensity is 520 nm. It is found that the color intensity is directly proportional to the concentration of urea in the given sample APPLICATIONS of Urea Assay Kit: • Direct Assays: level of urea determination in serum, plasma, urine, milk etc • Environment: determination of urea in waste water, soil etc. • Drug Discovery/Pharmacology: how the drugs behave on urea metabolism CONTENTS of Urea Assay Kit: 50 mL of Reagent A, 50 mL of Reagent B and 1 mL 50 mg/dL urea standard. Storage of Urea Assay Kit: at 4°C all components remain stable for 12 months. . Protocol of Urea Assay Kit: Preparation of Reagent: • All reagents are Equilibrate at room temperature • Sufficient Working Reagents are prepared for 96 well-plates • Reagent A and Reagent B, are combined with equal volumes • after the mixing of working reagent, mixer should be Used within 20 min Procedure: • Samples: Serum and plasma samples are directly assayed. Urine samples is diluted 5-fold with distilled water before assay • 5? µL of water (blank), samples and standard (50mg/dL) are transferred into a clear bottom well of 96-well plate. • 200 µL working reagent is added and then the plate is tapped lightly to mix properly • Incubation is done at room temperature for 10-20 min and optical density reading is taken at 470-550 nm .absorbance peak is reached at 520nm. Procedure using cuvette: • diluted samples are Prepared according to 96-well plate assay • Standard and diluted samples and 20 µL are transferred to the labeled tubes. • 1000 µL working reagent is added and then the plate is tapped lightly to mix properly • Allow it to nurture at room temperature for 10-20 min and optical density reading is taken at 520 nm On the other hand UREASE acts as an enzyme which catalyzes the urea hydrolysis and results in the formation of ammonia and carbon dioxide at 670nm. Urease Assay Kit of Bio Assay Systems provides a convenient, stable, sensitive and high-throughput adaptable method that helps to measure activity of urease in various samples. Urease Assay Kit is non-radioactive and contain 20mL assay buffer,12 mL reagent A and 6 mL of reagent B, 100 µL 50mM NH4Cl. All reagents are stored at 4oc and life of the shelf is six months after you receive the kit. Urease Assay PROCEDURE: • at first all the component are brought at room temperature and 495 µL BUFFER,5 µL 50mM NH4Cl are mixed to prepare 500 µL premix. • 10 µL urea is added to each well and then allow incubating for 10min at required temperature. • 100µL reagent A is added to each well and the plate is tapped to mix. After that 50 µL reagent B is added and again allows tapping so that it can mix well. • Incubation is allowed for 30min in the dark and reading of optical intensity is taken at 670nm. • For more information about Urease Assay Kit please visit http://www.bioassaysys.com
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