Products
Urea Nitrogen Content Assay Kit
- Catalog Number
FNK063
- Specification
120T/50S
- Species
Universal
- Application
Quantitative detection of urea nitrogen content in liquid samples such as tissues, cells, bacteria, serum (plasma), and culture media etc.
- Storage Conditions
Store in a dark place at 2-8°C
- Shelf Life
3 months
- Note
For research use only.
- Reagent Components
Name
Reagent Size
Storage
Instructions
Reagent 1
Powder x2 vial
Store at 2-8°C in dark
Completely dissolve each vial with 5mL distilled water before use(Use immediately after preparation, Store for one week at 2-8°C)
Reagent 2
Liquid 15mLx1 vial
Store at 2-8°C
-
Reagent 3
A Liquid
Liquid 3mLx1 vial
Store at 2-8°C in dark
Before use, add A Liquid into B Liquid to completely mix(Or mix A:B in a ratio of 1:4, Use immediately after preparation, Store for one week at 2-8°C)
B Liquid
Liquid 12mLx1 vial
Store at 2-8°C in dark
Reagent 4
Liquid 2mLx1 vial
Store at 2-8°C in dark
Before use, add 13mL distilled water to completely mix
Standard
Powder x1 vial
(10mg urea standard)
Store at 2-8°C
Before use, add 466mL distilled water to completely dissolve
(Yielding 10mg/mL urea nitrogen standard solution)
Preparation of Standard Diluent: Dilute 10 mg/mL nitrogen standard solution with distilled water to obtain standard diluent of 40, 30, 20, 10, 5, 2.5 µg/mL.
- Assay Principle
Urea nitrogen is the main end product in human protein metabolism, and consists of most non-protein nitrogens in the blood. Nitrogen content in serum urea is closely related to glomerular function, and can reflect the filtration function of glomerular in a certain degree. It's also one of the main factors for measuring renal function.
NH3-N produced by urease-induced urea hydrolysis can react with hypochlorite and phenol in strong alkaline medium, resulting in the water-soluble blue dye alpha-naphthol blue. The alpha-naphthol blue has special absorption peak at 630nm. The contend of urea nitrogen can be quantitatively measured via the change of absorption value.
- Additional Materials Required
1. Visible spectrophotometer / ELISA reader (630 nm)
2. Microglass cuvettes (10 mm light path) / 96-well plate
3. Mortar and pestle
4. Precision single and multichannel pipettes with clean disposable tips
5. Clean EP tubes
6. Distilled water
7. Benchtop centrifuge
8. Constant temperature water bath / incubator
- Sample Preparation (Change sample quantity and ratio according to pilot assay results)
Tissue: Process the sample at a ratio of tissue mass (g) to distilled water volume (mL) of 1: (5-10) (suggested to take 0.1 g tissue and add 1 mL distilled water), homogenize on ice, centrifuge at 12000 g for 15 min at 4°C, and collect the supernatant for measurement on ice.
Bacteria or cells: Collect bacteria or cells into centrifuge tubes, process the sample at a ratio of the number of bacteria or cells (10^4) to distilled water volume (mL) of (500-1000): 1 (suggested to add 5 million bacteria or cells to 1 mL distilled water), perform ice-bath sonication (300 W power, 3 s ultrasound, 7 s interval, total time 3 min), centrifuge at 12000 g for 15 min at 4°C, and collect the supernatant for measurement on ice.
Serum (plasma), culture media, and other liquid samples: Directly detect or dilute appropriately before detection.
- Assay Procedure
1. Preheat the spectrophotometer or ELISA reader for 30 min, set the wavelength to 630 nm, and zero with distilled water.
2. Sequentially add the following reagents into the centrifuge tube.
Reagent
Measurement Tube(μl)
Control Tube(μl)
Standard Tube(μl)
Blank Tube(μl)
Sample
30
30
-
-
Standard Dilution
-
-
30
-
Distilled Water
-
-
-
60
Reagent 1
60
-
60
60
Reagent 2
110
110
110
110
Thoroughly mix, incubate at 37°Cfor 10 minutes
Reagent 3
100
100
100
100
Reagent 4
100
100
100
100
Thoroughly mix, incubate at 37°Cfor 30 minutes
- Measurement of Absorption Value
Add 200μL reaction solution into the 96-well plate or microglass cuvettes to measure absorption value at 630nm, and then label as A Measurement, A Control, A Standard and A Blank respectively; Calculate ∆A Measurement = A Measurement - A Control, ∆A standard = A Standard - A Control. Note: Measure the blank tube once or twice. Set a control tube for each sample.
- Establishment of Standard Curve
Plot the standard curve based on the horizontal coordinate-axis X(using standard diluent concentration 40, 30, 20, 10, 5, 2.5 µg/mL) and the corresponding longitudinal coordinate-axis Y(using ∆A standard) to get the standard equation y=kx+b. Bring ∆A Measurement into the equation to get x(µg/mL).
- Calculation of Urea Nitrogen Content
1.Calculate According to Tissue Sample Quality
2.Calculate According to Tissue Protein Concentration
3.Calculate According to number of bacteria or cells
4.Calculate According to Liquid Volume
Notes: VB: total volume of pilot sample, 1mL; VL: total volume of liquid sample, mL; W: sample quality, g; Cpr: sample protein concentration, mg/mL; number of bacteria or cells: calculated by ten thousand
- Notes for Assay
1. A Measurement is beyond the linear range of standard absorption value: when higher than the highest value, it's suggested to properly dilute pilot sample and measure again; when lower than the lowest value, properly add sample volume and prepare the sample again for a remeasurement. Modify properly during calculation.
2.Complete the measurement within 1h after the staining. The colour of measurement tube and standard tube should be the same(blue). If the staining turns yellow or green, the concentration of NH3-N is higher. The pilot sample should be properly diluted and measured again. Modify properly during calculation.
3.To ensure accurate result and avoid reagent loss, please carefully read the manual before the measurement(following the printed version upon receipt). Check whether the reagent storage & preparation and operation steps are clear. Take 2-3 samples with significant differences to conduct the pilot assay. Please contact us if you are experiencing any problem during the assay.